Open Access

The ribosomal protein genes and Minute loci of Drosophila melanogaster

  • Steven J Marygold1Email author,
  • John Roote2,
  • Gunter Reuter3,
  • Andrew Lambertsson4,
  • Michael Ashburner2,
  • Gillian H Millburn2,
  • Paul M Harrison5,
  • Zhan Yu5,
  • Naoya Kenmochi6,
  • Thomas C Kaufman7,
  • Sally J Leevers1 and
  • Kevin R Cook7Email author
Genome Biology20078:R216

DOI: 10.1186/gb-2007-8-10-r216

Received: 17 June 2007

Accepted: 10 October 2007

Published: 10 October 2007

Abstract

Background

Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes.

Results

We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci.

Conclusion

This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome.

Background

Ribosomes are sophisticated macromolecular machines that catalyze cellular protein synthesis in all cells of all organisms. They have an ancient evolutionary origin and are essential for cell growth, proliferation and viability. Though larger and more complex in higher organisms, both the structure and function of ribosomes have been conserved throughout evolution. Genetic approaches in Drosophila melanogaster have shown that disrupting ribosome function can result in an array of fascinating dominant phenotypes [1, 2]. Despite this, there has so far been no comprehensive inventory of genes encoding ribosome components in this organism, nor any systematic effort to determine their mutant phenotypes.

All ribosomes comprise a set of ribosomal proteins (RPs) surrounding a catalytic core of ribosomal RNA (rRNA). Bacteria possess a single type of ribosome composed of three rRNA molecules and typically 54 RPs. All eukaryotic cells, in contrast, contain at least two distinct types of ribosomes: cytoplasmic ribosomes (cytoribosomes) and mitochondrial ribosomes (mitoribosomes). Cytoribosomes are found on the endoplasmic reticulum and in the aqueous cytoplasm. They translate all mRNAs produced from nuclear genes and perform the vast majority of cellular protein synthesis. Each cytoribosome contains four different rRNAs and 78-80 cytoplasmic RPs (CRPs). Mitoribosomes consist of only two rRNA molecules and up to 80 mitochondrial RPs (MRPs). They are located in the mitochondrial matrix and synthesize proteins involved in oxidative phosphorylation encoded by those few genes retained in the mitochondrial genome. A third unique type of eukaryotic ribosome is found within the plastids (for example, chloroplasts) of plant and various algal cells. In all cases, distinct small and large ribosomal subunits exist that join together during the translation initiation process to form mature ribosomes capable of protein synthesis. (See references [36] for general reviews of ribosomal structure and function.)

The protein components of ribosomes are interesting from several points of view. First, and most obviously, RPs play critical roles in ribosome assembly and function [7]. Second, several RPs perform important extra-ribosomal functions, including roles in DNA repair, transcriptional regulation and apoptosis [6, 8]. Third, misexpression of human CRP and MRP genes has been implicated in a wide spectrum of human syndromes and diseases, including Diamond-Blackfan anaemia [9], Turner syndrome [10], hearing loss [11] and cancer [12]. Fourth, mutations in the CRP genes of D. melanogaster are important tools for the study of growth, development and cell competition [2]. Finally, many RPs are conserved from bacteria to humans, so their peptide and nucleotide sequences are useful for studying phylogenetic relationships [13].

The first eukaryotic CRPs characterized in detail were isolated from the rat cytoribosome [3]. Individual proteins were separated by two-dimensional gel electrophoresis and named from their origin in the small (S) or large (L) subunit and their relative electrophoretic migration positions, for example, RPS9 or RPL28. Subsequent studies revealed that some protein spots contained non-ribosomal proteins or chemically modified versions of another CRP, and that some spots contained two co-migrating CRPs [3, 5]. Consequently, the nomenclature system used today contains numerical gaps as well as 'A' suffixes for those additional CRPs not resolved by the original electrophoresis (for example, RPL36A). Seventy-nine distinct mammalian CRPs are now acknowledged and their amino acid sequences and biochemical properties have been described [5, 14]. With the exception of RPLP1 and RPLP2, each of which forms homodimers in the cytoribosomal large subunit [15], all CRPs are present as single molecules in each cytoribosome [3].

Seventy-eight different mammalian MRPs have been described [6] and their individual amino acid sequences and biochemical properties have been determined [16, 17]. Although the nomenclature of MRPs was originally based on electrophoretic properties, the current system reflects homology between mammalian MRPs and their bacterial orthologs [18]. Thus, MRPS1 through MRPS21 are orthologous to Escherichia coli RPs S1-S21, while higher numbers have been assigned to the MRPs not found in bacteria. Gaps also exist in MRP numbering because a gap occurs in the bacterial enumeration or because there is no mammalian ortholog.

The RPs of D. melanogaster were first studied in the 1970s and early 1980s. Up to 78 individual CRPs were observed on two-dimensional gels [1931] and about 30 were purified and analyzed biochemically [32, 33]. A more recent characterization used mass spectrometry to identify 52 D. melanogaster CRPs [34], all of which are orthologous to known mammalian CRPs. The protein composition of Drosophila mitoribosomes has not been characterized biochemically to date.

CRPs and MRPs are encoded by the nuclear genome. Knowledge of the primary sequences of rat CRPs and bovine MRPs has led to the identification and mapping of the RP-encoding genes in many eukaryotic species [14]. Indeed, systematic analyses of whole RP gene sets have been described for several organisms, including Saccharomyces cerevisiae [35], Arabidopsis thaliana [36] and humans [3740]. However, the complete set of D. melanogaster CRP and MRP genes has not been previously documented or characterized.

Several D. melanogaster RP genes were initially identified by virtue of their dominant 'Minute' mutant phenotypes [2], which include prolonged development, low fertility and viability, altered body size and abnormally short, thin bristles on the adult body. All of these phenotypes may be explained by a cell-autonomous defect in protein biosynthesis: the production of each bristle, for example, requires a very high rate of protein synthesis in a single cell during a short developmental period. Merriam and colleagues reported the first unequivocal molecular link between a Minute locus and a CRP gene in 1985 [41]. Since then, 14 additional Minute loci have been definitively linked with distinct CRP genes [2, 4253]. However, there are at least 35 genetically validated Minute loci that have not yet been associated with a specific gene and there may be additional Minute genes to be discovered. Several investigators have hypothesized that all Minute loci encode protein components of ribosomes (reviewed in [2]). Whether this is truly the case and whether both CRP and MRP genes are associated with Minute phenotypes are open and intriguing questions.

Many Minute loci were originally identified from the phenotypes of flies heterozygous for a chromosomal deletion [54, 55] and all Minute point mutations studied in depth have been found to be loss-of-function alleles [2]. This indicates that Minute phenotypes can be attributed to genetic haploinsufficiency; that is, a single gene copy is not sufficient for normal development. (Note that X-linked mutations that cause Minute phenotypes in heterozygous females are lethal in hemizygous males.) The most popular explanation for the haploinsufficiency of Minute loci is that they correspond to RP genes and that RPs are required in equimolar amounts: halving the copy number of a single RP gene limits the availability of the encoded RP, thereby reducing the number of functional ribosomes that are assembled in the cell and impairing protein synthesis [2]. While this idea is consistent with the available data, there may be other explanations.

The reduced fertility and viability associated with many Minute loci makes the recovery of deletions uncovering them rather difficult - the mutant strains are too weak to maintain as stable heterozygous stocks. In fact, some Minute loci are known only from the phenotypes of transient aneuploids [54, 56]. This means that several chromosomal regions containing a Minute locus are not uncovered by current deletion collections [57]. This is frustrating for researchers because deletions are basic tools for mutational analysis and are widely used for mapping new mutations and identifying genetic modifiers. Efforts to maximize deletion coverage of the D. melanogaster genome would benefit from a systematic assessment of the relationship between RP genes and Minute loci. It would allow the isolation of deletions that flank haploinsufficient RP genes as closely as possible, or the design of transgenic constructs or chromosomal duplications to rescue the haploinsufficiency of deletions uncovering Minute genes.

Here, we report the systematic identification, naming and characterization of all the CRP and MRP genes of D. melanogaster. We have used this information, together with phenotypic data obtained from examining mutation and deficiency strains, to assess the correspondence between RP genes and Minute loci. We find that 66 of the 88 CRP genes identified are, or are very likely to be, haploinsufficient and associated with a Minute phenotype, whereas MRP genes and the remaining 22 CRP genes are not. Significantly, we show that all but one of the known Minute loci in the genome correspond to CRP genes - the single exception encodes a subunit of an essential translation initiation factor. Together, these results identify the majority of haploinsufficient loci in the D. melanogaster genome that significantly affect viability, fertility and/or external morphology, and also provide a mechanistic framework for understanding the Minute syndrome and the phenotypic effects of aneuploidy.

Results

Identification of D. melanogasterribosomal protein genes

In order to conduct an exhaustive survey of Drosophila CRP and MRP genes, we first performed a series of BLAST searches using human RP sequences as queries, because both CRPs and MRPs have been well-characterized in humans [5, 6]. Tables 1 and 2 list the genes we identified together with their cytological locations. Where necessary, D. melanogaster genes were named or renamed according to the standard metazoan RP gene nomenclature proposed by Wool and colleagues [5, 58, 59] and approved by the HUGO Gene Nomenclature Committee [18], whilst still conforming to FlyBase [60] conventions - that is, CRP genes are given an 'Rp' prefix and MRP genes have an 'mRp' prefix. The seven exceptions to this standard RP nomenclature are mostly genes originally named to reflect a mutant phenotype, for example, the string of pearls (sop) gene encodes RpS2 [61] and bonsai encodes mRpS15 [62, 63]. In these cases, the original gene symbol has been preserved, with the apposite RP symbol given as a synonym.
Table 1

The CRP genes of D. melanogaster

 

D. melanogaster gene

 

Human CRP

Symbol*

CG number

Location

BLAST E value

RPSA

sta/RpSA

CG14792

X: 2B1

3e-94

RPS2

sop/RpS2

CG5920

2L: 30E1

e-118

RPS3

RpS3

CG6779

3R: 94E13

e-106

RPS3A

RpS3A

CG2168

4: 101F1

e-100

RPS4

RpS4

CG11276

3L: 69F6

e-121

RPS5

RpS5a

CG8922

X: 15E5-7

1e-98

 

RpS5b

CG7014

3R: 88D6

3e-96

RPS6

RpS6

CG10944

X: 7C2

e-103

 

CG11386

CG11386

X: 7C2

2e-15

 

CG33222

CG33222

X: 7C2

2e-15

RPS7

RpS7

CG1883

3R: 99E2

1e-74

RPS8

RpS8

CG7808

3R: 99C4

1e-82

RPS9

RpS9

CG3395

3L: 67B11

8e-92

RPS10

RpS10a

CG12275

3R: 98A14

4e-43

 

RpS10b

CG14206

X: 18D3

8e-52

RPS11

RpS11

CG8857

2R: 48E8-9

1e-58

RPS12

RpS12

CG11271

3L: 69F5

2e-43

RPS13

RpS13

CG13389

2L: 29B2

2e-74

RPS14

RpS14a

CG1524

X: 7C6-7

3e-71

 

RpS14b

CG1527

X: 7C8

3e-71

RPS15

RpS15

CG8332

2R: 53C8

6e-62

RPS15A

RpS15Aa

CG2033

X: 11E11-12

2e-65

 

RpS15Ab

CG12324

2R: 47C1

6e-65

RPS16

RpS16

CG4046

2R: 58F1

2e-69

RPS17

RpS17

CG3922

3L: 67B5

5e-52

RPS18

RpS18

CG8900

2R: 56F11

2e-69

RPS19

RpS19a

CG4464

X: 14F4

5e-48

 

RpS19b

CG5338

3R: 95C13

1e-43

RPS20

RpS20

CG15693

3R: 93A1

7e-50

RPS21

oho23B/RpS21

CG2986

2L: 23B6

3e-30

RPS23

RpS23

CG8415

2R: 50E4

8e-70

RPS24

RpS24

CG3751

2R: 58F3

7e-55

RPS25

RpS25

CG6684

3R: 86D8

1e-38

RPS26

RpS26

CG10305

2L: 36F4

3e-47

RPS27

RpS27

CG10423

3R: 96C8

2e-39

RPS27A

RpS27A

CG5271

2L: 31E1

8e-80

RPS28

RpS28a

CG15527

3R: 99D2

1e-21

 

RpS28b

CG2998

X: 8E7

2e-23

 

RpS28-like

CG34182

2L: 30B3

1e-07

RPS29

RpS29

CG8495

3R: 85E8

5e-23

RPS30

RpS30

CG15697

3R: 93A2

6e-32

RPLP0

RpLP0

CG7490

3L: 79B2

e-122

 

RpLP0-like

CG1381

2R: 46E5-6

3e-10

RPLP1

RpLP1

CG4087

2L: 21C2

3e-34

RPLP2

RpLP2

CG4918

2R: 53C9

5e-32

RPL3

RpL3

CG4863

3R: 86D8

0.0

RPL4

RpL4

CG5502

3R: 98B6

e-141

RPL5

RpL5

CG17489

2L: h35/40B

e-120

RPL6

RpL6

CG11522

3R: 100C7

8e-58

RPL7

RpL7

CG4897

2L: 31B1

2e-75

 

RpL7-like

CG5317

2L: 33C1

3e-32

RPL7A

RpL7A

CG3314

X: 6B1

e-102

RPL8

RpL8

CG1263

3L: 62E7

e-119

RPL9

RpL9

CG6141

2L: 32C1

1e-66

RPL10

Qm/RpL10

CG17521

3L: h47/80A

7e-98

RPL10A

RpL10Aa

CG3843

3R: 88D10

3e-65

 

RpL10Ab

CG7283

3L: 68E1

3e-95

RPL11

RpL11

CG7726

2R: 56D7

6e-83

RPL12

RpL12

CG3195

2R: 60B7

7e-75

RPL13

RpL13

CG4651

2L: 30F3

1e-68

RPL13A

RpL13A

CG1475

3R: 83B6-7

3e-68

RPL14

RpL14

CG6253

3L: 66D8

2e-30

RPL15

RpL15

CG17420

3L: h50-52/80F

5e-90

RPL17

RpL17

CG3203

X: 6C10

5e-72

RPL18

RpL18

CG8615

3L: 65E9

3e-71

RPL18A

RpL18A

CG6510

2R: 54C3

3e-68

RPL19

RpL19

CG2746

2R: 60E11

4e-83

RPL21

RpL21

CG12775

2L: 40A-B

4e-66

RPL22

RpL22

CG7434

X: 1C4

4e-40

 

RpL22-like

CG9871

2R: 59D3

2e-24

RPL23

RpL23

CG3661

2R: 59B3

1e-68

RPL23A

RpL23A

CG7977

3L: 62A10

7e-52

RPL24

RpL24

CG9282

2L: 34B10

7e-55

 

RpL24-like

CG6764

3R: 86E5

8e-14

RPL26

RpL26

CG6846

3L: 75E4

1e-59

RPL27

RpL27

CG4759

3R: 96E9-10

1e-43

RPL27A

RpL27A

CG15442

2L: 24F3

5e-60

RPL28

RpL28

CG12740

3L: 63B14

3e-31

RPL29

RpL29

CG10071

2R: 57D8

8e-14

RPL30

RpL30

CG10652

2L: 37B9

3e-46

RPL31

RpL31

CG1821

2R: 45F5

5e-47

RPL32

RpL32

CG7939

3R: 99D3

6e-58

RPL34

RpL34a

CG6090

3R: 96F10

2e-29

 

RpL34b

CG9354

3R: 85D15

1e-29

RPL35

RpL35

CG4111

X: 5A11

2e-38

RPL35A

RpL35A

CG2099

3R: 83A4

1e-35

RPL36

RpL36

CG7622

X: 1B12

3e-33

RPL36A

RpL36A

CG7424

2L: 28D3

3e-43

RPL37

RpL37a

CG9091

X: 13B1

2e-39

 

RpL37b

CG9873

2R: 59C4

1e-31

RPL37A

RpL37A

CG5827

2L: 25C4

2e-38

RPL38

RpL38

CG18001

2R: h46/41C-E

2e-25

RPL39

RpL39

CG3997

2R: 60B7

3e-18

RPL40

RpL40

CG2960

2L: 24E1

2e-69

RPL41

RpL41

CG30425

2R: 60E5

8e-08

*Additional gene synonyms exist in most cases [60]. Bold font indicates CRP-like genes, putative pseudogenic fragments (CG11386 and CG33222) or the member of a duplicate gene pair that is expressed in a small number of tissues and/or at relatively low levels. Computed cytological position is given for euchromatic genes (Genome Release 5 [60]). The cytological and h-band locations for heterochromatic genes are based on data in reference [151] or estimated from images of in situ hybridizations of BACs to polytene chromosomes (RpL5 and RpL21) [152]. The h-band location of RpL15 was provided by B Honda (personal communication). Expect (E) value obtained from a BLASTp search of the D. melanogaster annotated proteome (Genome Release 5.1) with human RefSeq CRP sequences. (E values corresponding to RpL15 and RpS28-like were obtained from a BLAST search using Release 5.3.) Where multiple protein isoforms exist, the highest scoring hit is given.

Table 2

The MRP genes of D. melanogaster

 

D. melanogaster gene

 

Human MRP

Symbol*

CG number

Location

BLAST E value

MRPS2

mRpS2

CG2937

2L: 25B1

1e-69

MRPS5

mRpS5

CG40049

3L: h47/80A-B

4e-63

MRPS6

mRpS6

CG15016

3L: 64B2

2e-20

MRPS7

mRpS7

CG5108

2L: 31D11

6e-38

MRPS9

mRpS9

CG2957

3R: 84E4

8e-81

MRPS10

mRpS10

CG4247

3R: 88E3

1e-33

MRPS11

mRpS11

CG5184

3R: 89E11

8e-26

MRPS12

tko/mRpS12

CG7925

X: 3A3

1e-33

MRPS14

mRpS14

CG32531

X: 18C7

1e-35

MRPS15

bonsai/mRpS15

CG4207

2R: 58F3

1e-15

MRPS16

mRpS16

CG8338

2R: 50E1

2e-24

MRPS17

mRpS17

CG4326

2R: 60C1

2e-14

MRPS18A

mRpS18A

CG31450

3R: 85A3

8e-13

MRPS18B

mRpS18B

CG10757

2L: 38B6

4e-33

MRPS18C

mRpS18C

CG9688

3R: 99F4

7e-23

MRPS21

mRpS21

CG32854

3R: 87E8

3e-22

MRPS22

mRpS22

CG12261

3R: 98B6

3e-38

MRPS23

mRpS23

CG31842

2L: 34D6

2e-20

MRPS24

mRpS24

CG13608

3R: 95E6

2e-31

MRPS25

mRpS25

CG14413

X: 12F1

1e-49

MRPS26

mRpS26

CG7354

3L: 75B9

3e-11

MRPS27

NA

NA

NA

No hit

MRPS28

mRpS28

CG5497

2R: 55E2

1e-27

DAP3/MRPS29

mRpS29

CG3633

2R: 58E1

7e-72

MRPS30

mRpS30

CG8470

X: 13E18

3e-21

MRPS31

mRpS31

CG5904

3L: 72C2

5e-35

MRPS33

mRpS33

CG10406

3R: 89B16

3e-30

MRPS34

mRpS34

CG13037

3L: 72E1-2

8e-06

MRPS35

mRpS35

CG2101

3L: 62F4

2e-72

MRPS36

NA

NA

NA

No hit

MRPL1

mRpL1

CG7494

3R: 84F9-10

8e-25

MRPL2

mRpL2

CG7636

3L: 68A7

2e-56

MRPL3

mRpL3

CG8288

X: 13E14

5e-52

MRPL4

mRpL4

CG5818

2L: 35F1

6e-70

MRPL9

mRpL9

CG31478

3R: 88F1

6e-17

MRPL10

mRpL10

CG11488

2L: 21B4

8e-20

MRPL11

mRpL11

CG3351

3R: 88C3

4e-29

MRPL12

mRpL12

CG5012

3L: 66E5

2e-13

MRPL13

mRpL13

CG10603

2L: 37B1

2e-47

MRPL14

mRpL14

CG14048

X: 3A1

3e-31

MRPL15

mRpL15

CG5219

3L: 77C3

4e-74

MRPL16

mRpL16

CG3109

X: 2B14

3e-58

MRPL17

mRpL17

CG13880

3L: 61B3

8e-21

MRPL18

mRpL18

CG12373

2R: 49C2

3e-22

MRPL19

mRpL19

CG8039

3R: 85A5

1e-56

MRPL20

mRpL20

CG11258

3L: 69F5

8e-21

MRPL21

mRpL21

CG9730

3L: 76A3

2e-19

MRPL22

mRpL22

CG4742

X: 15A7-8

4e-41

MRPL23

mRpL23

CG1320

3L: 62D7

4e-28

MRPL24

mRpL24

CG8849

2L: 25B4

2e-45

MRPL27

mRpL27

CG33002

2L: 24F3

2e-10

MRPL28

mRpL28

CG3782

2L: 25B5

5e-27

MRPL30

mRpL30

CG7038

X: 4C11

3e-15

MRPL32

mRpL32

CG12220

3R: 100B8

6e-15

MRPL33

mRpL33

CG3712

X: 4B6

5e-08

MRPL34

mRpL34

CG34147

2R: 52E4

1e-07

MRPL35

mRpL35

CG13410

3R: 94A1

4e-25

MRPL36

mRpL36

CG18767

3L: 66B7

2e-09

MRPL37

mRpL37

CG6547

3R: 86C6

4e-16

MRPL38

mRpL38

CG15871

X: 12E5

2e-61

MRPL39

mRpL39

CG17166

3L: 71B1

2e-57

MRPL40

mRpL40

CG5242

3R: 86E4

6e-13

MRPL41

mRpL41

CG12954

2R: 51E7

2e-11

MRPL42

mRpL42

CG12921

2R: 46E1

1e-11

MRPL43

mRpL43

CG5479

2R: 59F6

1e-25

MRPL44

mRpL44

CG2109

3R: 83A4

6e-39

MRPL45

mRpL45

CG6949

3R: 94B6

4e-61

MRPL46

mRpL46

CG13922

3L: 62B4

5e-39

MRPL47

Rlc1/mRpL47

CG9378

3R: 85D19

1e-35

MRPL48

mRpL48

CG17642

2L: 22B1

4e-15

MRPL49

mRpL49

CG4647

X: 11D1

9e-22

MRPL50

mRpL50

CG8612

3L: 65E9

6e-09

MRPL51

mRpL51

CG13098

2L: 29D4

2e-13

MRPL52

mRpL52

CG1577

2R: 43E9

2e-14

MRPL53

mRpL53

CG30481

2R: 50C16

3e-06

MRPL54

mRpL54

CG9353

2R: 57B16

2e-18

MRPL55

mRpL55

CG14283

3R: 91F1

3e-17

LACTB/MRPL56

NA

NA

NA

No hit

*Additional gene synonyms exist in many cases [60]. Computed cytological position is given for euchromatic genes (Genome Release 5 [60]). mRpS5 h-band location provided by C Smith (DHGP, personal communication) and cytological position inferred from reference [151]. Expect (E) value obtained from a BLASTp search of the D. melanogaster annotated proteome (Genome Release 5.1) with human RefSeq MRP sequences. Where multiple protein isoforms exist, the highest scoring hit is given.

Cytoplasmic ribosomal protein genes

We identified 88 genes that encode a total of 79 different CRPs (Table 1). Thus, the D. melanogaster proteome contains orthologs of all 79 mammalian CRPs (32 small subunit and 47 large subunit proteins). While the majority of CRPs are encoded by single genes, nine are encoded by two distinct genes. In addition, we identified another five genes predicted to encode proteins with significantly lower similarity to human CRPs, which we term 'CRP-like' genes. Two fragments of the RpS6 gene were also identified. (The list of 88 CRP genes presented by Cherry et al. [64] originated from an earlier report of our results to FlyBase (MA and SJM, FBrf0178764). These authors also list five CRP-like genes from our original report, but two of these have been eliminated and two additional CRP-like genes have been added in the current analysis.)

The deduced characteristics of D. melanogaster and human CRPs are compared in Additional data file 1. As might be expected, the amino acid identity between the CRPs of the two species is very high (average of 69% with a range of 27-98%, excluding the CRP-like proteins) and the predicted molecular weights and isoelectric points of the homologous proteins are very similar. However, several D. melanogaster proteins (RpL14, RpL22, RpL23A, RpL29, RpL34a, RpL34b, RpL35A) have significantly lower overall identity and different molecular weights owing to terminal deletions or extensions (data not shown; also see [65]). (If these seven proteins are discounted, the average identity of fly and human CRPs increases to 72% with a range of 43-98%.) Similar to humans and other species, there are very few acidic CRPs in D. melanogaster: only six proteins (RpSA, RpS12, RpS21, RpLP0, RpLP1 and RpLP2) have isoelectric points less than pH 7. (Note that RpS21 is an acidic protein, whereas its human counterpart is basic.) As in other eukaryotes, RpS27A and RpL40 are carboxyl extensions of ubiquitin [6669], and, as in other animals, RpS30 is fused to a ubiquitin-like sequence. From these gross characterizations of component proteins, it appears that the fly cytoribosome differs only slightly from its human counterpart and is essentially the same as other eukaryotic cytoribosomes.

Previous biochemical analyses estimated that the D. melanogaster cytoribosome contains up to 78 CRPs [29]. This figure compares very well to the 79 different CRPs predicted by our orthology analysis (Table 1). Unfortunately, very few of the CRPs identified in the 1970s and 1980s were characterized to the level of amino acid sequence, so their correspondences to CRP genes are generally unknown, though there are a few exceptions (see references [7073]). We have been unable, therefore, to correlate the CRPs identified in these earlier studies with those encoded by the CRP genes identified in this study. In contrast, our CRP inventory certainly does contain all 52 CRPs identified by the recent biochemical analysis of D. melanogaster cytoribosomes by Alonso and Santarén [34].

Mitochondrial ribosomal protein genes

We identified 75 D. melanogaster genes encoding proteins of the mitoribosome (28 in the small subunit and 47 in the large subunit) by orthology to human MRPs (Table 2). These data complement and extend previous analyses of homology between human and D. melanogaster MRPs [16, 17]. As in these previous studies, genes encoding orthologs of three human MRPs (MRPS27, MRPS36 and LACTB/MRPL56) were not found.

The MRPs of humans and D. melanogaster are much more divergent than are their CRPs: MRPs have an average identity of only 34% (with a range of 15-57%) and several homologous pairs differ markedly in their sizes and isoelectric points (Additional data file 2). Indeed, it is known that the mitoribosome is a rapidly evolving structure whose composition varies among eukaryotic organisms [6]. It is quite possible that there are proteins in Drosophila mitoribosomes that are not found in their human counterparts and these will have been missed by our orthology analysis - a definitive inventory will require biochemical characterization of the fly mitoribosome. As in mammals, three distinct genes encode three different isoforms of MRPS18 (Table 2); it is thought that each mitoribosome contains a single MRPS18 protein and that mitoribosomes may, therefore, be heterogeneous in composition [6].

Duplicate cytoplasmic ribosomal protein genes

Of the 79 different CRPs of D. melanogaster, 9 are encoded by two distinct genes (Table 1). These are distinguished by a lowercase 'a' or 'b' suffix to the gene symbol. (The lowercase 'a' should not be confused with the uppercase 'A' suffix used in the standard CRP nomenclature; for example, RpL37a and RpL37A are different genes that encode different proteins.) Six of these gene pairs encode proteins of the small ribosomal subunit and the other three encode large subunit proteins. In humans, each CRP is typically encoded by a single, functional gene [37, 74], but thousands of nonfunctional CRP pseudogenes are known to exist [75]. We therefore investigated the evolutionary origin, sequence conservation and expression profile of the duplicate D. melanogaster CRP genes in order to assess whether both members of each pair are likely to be functional (Table 3 and Figure 1).
Table 3

Analysis of duplicate CRP genes and CRP-like genes

 

Gene

K A /K S b

cDNA clones

 
 

Symbola

Location

Comments

Pair-wisec

Branch-specificd

Totale

% testisf

% Amino acid identityg

RpS5

RpS5a

X: 15E5-7

-

0.07

0.09

133

1

78

 

RpS5b

3R: 88D6

-

 

0.09

60

7

 

RpS6

RpS6

X: 7C2

-

NA

NA

150

5

33h

 

CG11386

X: 7C2

CG11386 and CG33222 are tandem repeats of the third exon and flanking regions of RpS6

 

NA

0

0

 
 

CG33222

X: 7C2

  

NA

3

0

 

RpS10

RpS10a

3R: 98A14

Lacks introns; likely retrogene

0.10

0.15

1

0

73

 

RpS10b

X: 18D3

-

 

0.01

156

0

 

RpS14

RpS14a

X: 7C6-7

RpS14a and RpS14b share identical gene structures

NA

NA

154

2

100

 

RpS14b

X: 7C8

  

NA

12

0

 

RpS15A

RpS15Aa

X: 11E11-12

-

0.16

0.00

150

1

98

 

RpS15Ab

2R: 47C1

Lacks introns; likely retrogene

 

NA

67

1

 

RpS19

RpS19a

X: 14F4

-

0.09

0.01

134

1

65

 

RpS19b

3R: 95C13

-

 

0.08

1

100

 

RpS28

RpS28a

3R: 99D2

Lacks introns; likely retrogene

0.05

0.06

0

0

82

 

RpS28b

X: 8E7

-

 

0.00

131

1

 
 

RpS28-like

2L: 30B3

-

NAi

NA

1

0

36/37j

RpLP0

RpLP0

3L: 79B2

-

NA

NA

157

2

18

 

RpLP0-like

2R: 46E5-6

Present in all eukaryotes

 

NA

11

0

 

RpL7

RpL7

2L: 31B1

-

NA

NA

168

4

30

 

RpL7-like

2L: 33C1

-

 

NA

32

0

 

RpL10A

RpL10Aa

3R: 88D10

Lacks introns; likely retrogene

0.05

0.06

7

71

64

 

RpL10Ab

3L: 68E1

-

 

0.02

160

4

 

RpL22

RpL22

X: 1C4

-

NA

NA

145

2

38

 

RpL22-like

2R: 59D3

-

 

NA

5

60

 

RpL24

RpL24

2L: 34B10

-

NA

NA

162

3

23

 

RpL24-like

3R: 86E5

Present in all eukaryotes

 

NA

34

3

 

RpL34

RpL34a

3R: 96F10

RpL34a and RpL34b share identical gene structures

0.04

0.09

56

4

78

 

RpL34b

3R: 85D15

  

0.04

132

2

 

RpL37

RpL37a

X: 13B1

-

0.01

NA

158

3

72

 

RpL37b

2R: 59C4

Lacks introns; likely retrogene

 

0.04

3

0k

 

aBold font indicates CRP-like genes, putative pseudogenic fragments (CG11386 and CG33222) or the member of a duplicate gene pair that is expressed in a small number of tissues and/or at relatively low levels. bK A /K S calculations are not applicable (NA) to highly diverged sequences or cases where the numbers of both synonymous and nonsynonymous substitutions are very small (<5). cCalculated for each D. melanogaster CRP gene pair using maximum likelihood analysis. Values for pairwise comparisons are shown on the first row of each pair. dBranch-specific score in a three-way maximum likelihood tree including D. pseudoobscura orthologs. A four-way tree was used for RpS19 sequences. eTotal number of cDNA clones (excluding those from cultured cell lines) given in FlyBase [60] (April 2007). RpS28-like cDNA evidence from L Crosby (personal communication). fPercentage of cDNA clones from adult testis cDNA libraries (AT, UT and BS), rounded to the nearest integer. gIdentity between proteins across their whole length. Values for pairwise comparisons are shown on the first row of each pair. hIdentity between RpS6 and the CG11386 or CG33222 protein. If CG11386 or CG33222 were used as alternative third exons of RpS6, the protein encoded would be 60% identical to the conventional RpS6 (see text for details). iRpS28-like is too highly diverged from both RpS28a and RpS28b for a pair-wise K A /K S calculation to be applicable. jIdentity between the RpS28-like protein and RpS28a/RpS28b. kThere is experimental evidence that RpL37b expression is enriched in adult testis [79].

https://static-content.springer.com/image/art%3A10.1186%2Fgb-2007-8-10-r216/MediaObjects/13059_2007_Article_1696_Fig1_HTML.jpg
Figure 1

Evolution of D. melanogaster CRP gene duplicates and CRP-like genes. The likely pattern of emergence of CRP duplicate genes with restricted expression (blue), CRP-like genes (green) and CRP pseudogenic fragments (brown) in the lineage leading to D. melanogaster is shown. RpL34b is shown in black text: this is the only case where the newly emerged duplicate gene (RpL34b), rather than precursor gene (RpL34a), acts as the principal gene copy. The relative placement of CG11386 and CG33222 is consistent with the model presented by Stewart and Denell [86]. The dendrogram is based on that given in reference [140], in which the relationships among the Drosophilidae are taken from [149]; note that the branch lengths do not accurately reflect evolutionary time.

In five cases, one member of the gene pair lacks introns (RpS10a, RpS15Ab, RpS28a, RpL10Aa and RpL37b) while the other member does not. These five intronless genes are likely to have arisen by retrotransposition; that is, generated via reverse transcription of mRNA from the precursor gene followed by insertion into a new genomic location. In contrast, the RpS5, RpS19 and RpL34 duplicates arose through gene transposition events as both members of each pair retain introns. The RpL34 duplication occurred through an intrachromosomal transposition on chromosome arm 3R, and RpL34a and RpL34b have retained almost identical gene structures. In contrast, the RpS5 and RpS19 duplications involved interchromosomal transposition events that must have been followed by extensive gene remodeling as the intron-exon structures differ within each pair. Finally, RpS14a and RpS14b probably arose via unequal exchange: these paralogs are situated adjacent to each other as a tandem duplication on the X chromosome, share identical intron-exon structures and encode identical proteins [76]. All nine duplicate genes appear to have arisen within the Drosophilidae, albeit at different stages in the lineage leading to D. melanogaster (Figure 1).

Neither member of these 9 CRP gene pairs contains a nonsense mutation in the protein-coding region (data not shown), indicating that all 18 genes are potentially functional. Moreover, the low ratio of nonsynonymous to synonymous substitutions (K A /K S ) between the members of each gene pair suggests that there are selective constraints on their protein-coding regions (Table 3; a K A /K S ratio significantly lower than 0.5 indicates functional constraints on both genes). Branch-specific K A /K S values further indicate that the putatively retrotransposed genes have been under overall purifying selection since their formation. Together, these data argue that none of these duplicate genes are nonfunctional pseudogenes, which is consistent with a previous analysis [77]. Indeed, the recovery of multiple cDNA clones for the majority (15/18) of these duplicate genes supports their expression in vivo (Table 3).

Although none of these CRP gene duplicates appear to be pseudogenes, it is evident that one member of each pair - the one with higher similarity to its human ortholog, where this difference exists (Table 1 and Additional data file 1) - is expressed at a significantly higher level and, in some cases, in a wider array of tissues than the other. This suggests that one gene of the pair produces the majority of each CRP in most cells, while the other gene has a more restricted expression pattern and, perhaps, a specialized function (indicated by bold font in Tables 1 and 3). In eight of the nine duplication events, the 'younger' gene copy has adopted the lower expression level or more restricted expression pattern; the RpL34 gene pair is exceptional in this regard (Figure 1 and Table 3). The expression of RpS5b, RpS19b, RpL10Aa and RpL37b appears enriched in the adult testis, suggesting the existence of testis-specific CRPs and a testis-specific cytoribosome (Table 3). Significantly, three of these genes (RpS5b, RpS19b and RpL37b), together with RpS10a, RpS15Ab and RpS28a, are autosomal copies of X-linked genes. These duplication events are consistent with previous studies reporting that genes with male-biased expression are predominantly autosomal [78], and that retrotransposed genes in D. melanogaster have preferentially retrotransposed from the X chromosome onto autosomes [79]. It is possible that these autosomal duplicates enable CRP expression in male germline cells, where it is hypothesized that X chromosome inactivation occurs during spermatogenesis [80]. Similarly, in humans, RPS4Y is a Y-linked duplicate of the X-linked RPS4 gene [10] and RPL10L, RPL36AL and RPL39L are autosomal retrogene copies of X-linked progenitors [74]. It is worth noting that expression of D. melanogaster RpS5b, RpS10a and RpS19b is also enriched in the germline cells of embryonic gonads [81] and/or stem cells of adult ovaries [82]. These findings suggest a germline-specific role, rather than a testis-specific role, for these CRP gene duplicates.

To conclude, the 'principal' CRPs of D. melanogaster - those that are expressed at high levels in most cells - are each encoded by single genes.

Cytoplasmic ribosomal protein-like genes

We identified five D. melanogaster 'CRP-like' genes that encode proteins with significantly lower identity to human CRPs than those described above. These are RpS28-like, RpLP0-like, RpL7-like, RpL22-like and RpL24-like (shown in bold font in Tables 1 and 3). Of these, RpLP0-like and RpL24-like show the most divergence from their cognate proteins, RpLP0 and RpL24. Consistent with this, RpLP0-like and RpL24-like have ancient evolutionary origins, while RpL7-like, RpL22-like and RpS28-like arose more recently within the Diptera (Figure 1).

cDNA evidence indicates that all five of these CRP-like genes are expressed in vivo, albeit at far lower levels than their cognate genes (Table 3). The evolutionary conservation of RpLP0-like and RpL24-like suggests they have important cellular functions. Indeed, the yeast ortholog of RpL24-like is found in pre-ribosomal complexes where it is thought to function in large subunit biogenesis [83]. It remains to be seen whether the other CRP-like proteins have similar functions. Interestingly, the RpL22 gene is X-linked and expressed ubiquitously, whereas RpL22-like is an autosomal gene that is expressed predominantly in germline cells [81, 82, 84, 85]. This suggests that RpL22-like may have a specialized role in the germline, and perhaps within germline-specific cytoribosomes, as proposed above for some of the CRP duplicates.

CG11386 and CG33222 are 99% identical in DNA sequence and are tandem repeats of the third exon and flanking regions of the RpS6 gene. They likely arose via two sequential unequal crossover events [86]; the first occurring after the evolutionary split of the melanogaster subgroup, and the second being specific to D. melanogaster (Figure 1). Gene prediction algorithms suggest that CG11386 and CG33222 are distinct genes encoding identical amino-terminally truncated versions of RpS6 [87]; however, such proteins would lack critical functional domains and would probably be nonfunctional. In a different scenario, CG11386 and/or CG33222 could serve as alternative third exons of the RpS6 gene: the proteins produced would be full-length, but would differ substantially in their carboxy-terminal two-thirds from the RpS6 generated by using the conventional third exon [86]. There is, however, no direct evidence that such alternative transcripts are made. Indeed, only three cDNA clones suggest that CG11386 or CG33222 are expressed at all (Table 3). We have tentatively classified CG11386 and CG33222 as nonfunctional pseudogenic fragments.

Chromosomal distribution of ribosomal protein genes

As has been found for other eukaryotes [35, 36, 38, 39], the RP genes of D. melanogaster are distributed across the entire genome (Figure 2). Some RP genes are tightly linked to other RP genes and, while this posed challenges for determining the phenotypes associated with individual genes (see below), we have no evidence that this distribution has functional consequences or that closely linked RP genes are transcriptionally co-regulated. Five RP genes (RpL5, Qm/RpL10, RpL15, RpL38, and mRpS5) are located within heterochromatic regions, as are certain human MRP genes [38] and some Arabidopsis thaliana CRP genes [36]. As heterochromatin is generally associated with the silencing of gene expression [88], the regulation of these genes must have adapted to the heterochromatic environment in order for the encoded proteins to be expressed at sufficiently high levels to meet the demand for ribosome synthesis in the cell [89].
https://static-content.springer.com/image/art%3A10.1186%2Fgb-2007-8-10-r216/MediaObjects/13059_2007_Article_1696_Fig2_HTML.jpg
Figure 2

Chromosomal map of the RP genes of D. melanogaster. RP genes are depicted on a physical map of the genome (Release 5) [60]. Genes encoded on the positive and negative strands are shown above and below the chromosome, respectively. (The orientation of RpL15 is not known and its position below the chromosome is arbitrary.) Chromosomes are divided into cytological bands as determined from sequence-to-cytogenetic band correspondence tables [150]. Minute genes are boxed as described in the key.

Ribosomal protein gene haploinsufficiency and the Minute syndrome

Classical genetic studies have defined more than fifty regions of the D. melanogaster genome that are haploinsufficient and associated with the dominant phenotypes of prolonged development and short, thin bristles - the Minute loci [2] (Figure 3). To date, only fifteen Minute loci have been tied unequivocally to molecularly defined genes and all of these encode RPs (reviewed in reference [2]; also see references [4853]). It has not been clear, however, if all Minute loci correspond to RP genes, or whether Minute loci may correspond to both CRP and MRP genes. We have conducted a new survey of Minute loci in the D. melanogaster genome which, combined with our RP gene inventory, has now allowed us to assess these relationships systematically.
https://static-content.springer.com/image/art%3A10.1186%2Fgb-2007-8-10-r216/MediaObjects/13059_2007_Article_1696_Fig3_HTML.jpg
Figure 3

The Minute bristle phenotype. Minute flies have shorter and thinner bristles than wild type flies. This is most clearly seen by comparing the scutellar bristles, indicated here by the arrows and pseudocoloring. (a, a') Wild type. (b, b') RpS131 heterozygotes. (c, c') RpL141 heterozygotes.

Recent large-scale projects have provided a wealth of new genetic reagents that enable the mapping of Minute loci with a precision unavailable only a few years ago. Hundreds of new deletions with molecularly defined breakpoints have been provided by the efforts of the DrosDel consortium [90, 91], Exelixis, Inc. [92], and the Bloomington Drosophila Stock Center [92]. When combined with older deletions characterized primarily through polytene chromosome cytology, these deletions have increased euchromatic genome coverage to 96-97%. In addition, transposable element insertions now exist within 0.5 kb of 57% of all genes (R Levis, personal communication), largely through the efforts of the Drosophila Gene Disruption Project [93] and Exelixis, Inc. [94]. We used these resources to conduct a genome-wide search for Minute loci. In so doing, we considered the characteristic Minute bristle phenotype (Figure 3) to be diagnostic of the Minute syndrome; we did not methodically evaluate more subtle Minute traits, such as slower development, or traits observed in only a subset of Minute mutants, such as impaired fecundity, reduced viability or altered body size. By combining our observations with information gleaned from published studies, we have identified 61 distinct Minute loci. Many of these correlate with Minute loci described previously (Additional data file 3), though our work has often refined their map positions. Significantly, six Minute loci (M(2)31E, M(2)34BC, M(2)45F, M(2)50E, M(3)93A and M(3)98B) are reported here for the first time. We also found four instances (M(2)31A, M(2)53, M(2)58F and M(3)67C) where a single Minute locus characterized by previous aneuploidy analyses actually comprises two separable, closely linked Minute genes. As we have inferred the existence of four additional Minute loci from patterns of deletion coverage (described below), we conclude that there are 65 distinct Minute loci in the D. melanogaster genome.

We were able to demonstrate definitively that a particular Minute locus corresponds to a specific RP gene when a Minute bristle phenotype was observed in one or more of the following situations: flies heterozygous for a molecularly characterized mutation in a RP gene (for example, M(2)36F/RpS26); flies heterozygous for a chromosomal deletion when the Minute phenotype could be mapped unambiguously to a single RP gene with deletion breakpoints (for example, M(2)25C/RpL37A); or flies heterozygous for a chromosomal deletion when the Minute phenotype could be rescued by a specific RP transgene (for example, M(3)99D/RpL32). We found that there are 26 unequivocally Minute CRP genes by these criteria (Additional data file 4; summarized in Table 4). In contrast, no MRP or CRP-like genes were definitively demonstrated to be Minute genes.
Table 4

CRP gene haploinsufficiency

CRP genea

Genetic analysisb

    

Symbol

Location

CRP gene mutationsc, d

Deletions removing a single CRP genec, e

Other evidence

Is the CRP gene a Minute?f

No. of candidate Minute genesg

Minute synonymh

Referencei

X chromosome

        

   RpL36

1B12

 

M

 

Yes

 

M(1)1B

[53]

   RpL22

1C4

+

+

 

No

   

   sta/RpSA

2B1

+

+

 

No

   

   RpL35

5A11

  

Lies in a gap in deletion coverage. 5A6-13 aneuploids were Minute (Merriam et al. [55])

Likely

33

M(1)5A

 

   RpL7A

6B1

 

M

 

Likely

2

M(1)5D6A

 

   RpL17

6C10

  

Lies in a gap in deletion coverage

Likely

12

New?

 

   RpS6

7C2

M

M

 

Yes

 

M(1)7BC and M(1)7C

[2]

   RpS14a

7C6-7

  

A deletion that removes RpS14a and RpS14b is not Minute [95]

No

   

   RpS14b

7C8

  

A deletion that removes RpS14a and RpS14b is not Minute [95]

No

   

   RpS28b

8E7

  

Lies in a gap in deletion coverage. A Minute mutation was mapped to 8D8-9A2 [153]

Likely

17

M(1)8F

 

   RpS15Aa

11E11-12

 

M

 

Likely

8

M(1)11F

 

   RpL37a

13B1

  

Lies in a gap in deletion coverage. 12F6-13B6 aneuploids were Minute (Merriam et al. [55])

Likely

2

M(1)13A

 

   RpS19a

14F4

 

M

 

Likely

14

M(1)14E

 

   RpS5a

15E5-7

M

 

Lies in a gap in deletion coverage

Yes

 

M(1)15D

[44]

   RpS10b

18D3

M

M

 

Yes

 

M(1)18C

 

Chromosome arm 2L

        

   RpLP1

21C2

M

M

 

Yes

 

M(2)21C

[50]

   oho23B/RpS21

23B6

M

M

 

Yes

 

M(2)23B

[49]

   RpL40

24E1

 

M

 

Likely

4

M(2)24D

 

   RpL27A

24F3

M

M

 

Yes

 

M(2)24F

 

   RpL37A

25C4

 

M

The interval between flanking non-Minute deletions contains only RpL37A

Yes

 

M(2)25C

 

   RpL36A

28D3

 

M

 

Likely

5

M(2)28DE*

 

   RpS13

29B2

M

M

 

Yes

 

M(2)29B*

[45]

   sop/RpS2

30E1

+

+

 

No

   

   RpL13

30F3

 

M

The interval between flanking non-Minute deletions contains only RpL13

Yes

 

M(2)31A

 

   RpL7

31B1

 

M

 

Likely

2

M(2)31A

 

   RpS27A

31E1

 

M

 

Likely

6

M(2)31E*

 

   RpL9

32C1

M

M

 

Yes

 

M(2)32D

[46]

   RpL24

34B10

 

M

 

Likely

8

M(2)34BC*

 

   RpS26

36F4

M

M

 

Yes

 

M(2)36F

 

   RpL30

37B9

+

+

 

No

   

   RpL21

40A-B

 

M

 

Likely

10

M(2)39F

 

   RpL5

40B

M

M

 

Yes

 

M(2)40B*

[51]

Chromosome arm 2R

        

   RpL38

41C-E

M

M

 

Yes

 

M(2)41A

[51]

   RpL31

45F5

M

 

Lies in a gap in deletion coverage

Yes

 

M(2)45F*

 

   RpS15Ab

47C1

 

+

 

No

   

   RpS11

48E8-9

M

 

Lies in a gap in deletion coverage

Yes

 

M(2)48E*

 

   RpS23

50E4

 

M

 

Likely

2

M(2)50E*

 

   RpS15

53C8

  

M(2)531 Minute phenotype rescued by duplication of 51F-54A [154] but not by a RpLP2 transgene [155]

Likely

16

M(2)53

 

   RpLP2

53C9

 

M

 

Likely

5

M(2)53

 

   RpL18A

54C3

  

Lies in a gap in deletion coverage

Likely

16

New?

 

   RpL11

56D7

  

Lies in a gap in deletion coverage. 56C-D aneuploids were Minute [55]

Likely

3

M(2)56CD

 

   RpS18

56F11

 

M

 

Likely

2

M(2)56F

 

   RpL29

57D8

 

+

 

No

   

   RpS16

58F1

  

Deletions that remove both RpS16 and RpS24 are Minute. The Minute mutations M(2)58F1 and RpS24SH2053 complement, suggesting RpS16 is a Minute gene

Likely

25

M(2)58F

 

   RpS24

58F3

M

 

Deletions that remove both RpS16 and RpS24 are Minute

Yes

 

M(2)58F

 

   RpL23

59B3

 

+

 

No

   

   RpL37b

59C4

 

+

 

No

   

   RpL12

60B7

  

RpL12 and RpL39 lie in the same gap in deletion coverage. 60B3-10 aneuploids were Minute [156]

Likely

9

M(2)60B

 

   RpL39

60B7

  

RpL12 and RpL39 lie in the same gap in deletion coverage. 60B3-10 aneuploids were Minute [156]

Likely

9

M(2)60B

 

   RpL41

60E5

+

+

 

No

   

   RpL19

60E11

M

 

A deletion that removes RpL19 and RpL41 is Minute

Yes

 

M(2)60E

[42]

Chromosome arm 3L

        

   RpL23A

62A10

 

M

 

Likely

8

M(3)62A

 

   RpL8

62E7

  

Lies in a gap in deletion coverage containing only RpL8. 62E-63A aneuploids were Minute [54]

Yes

 

M(3)62F

 

   RpL28

63B14

 

M

 

Likely

10

M(3)63B

 

   RpL18

65E9

 

M

 

Likely

8

M(3)65F

 

   RpL14

66D8

M

 

Lies in a gap in deletion coverage

Yes

 

M(3)66D

[47]

   RpS17

67B5

  

A deletion removing both RpS17 and RpS9 is Minute. The unsequenced Minute mutations RpS174 and RpS176 complement the Minute mutation RpS9EP3299, suggesting RpS17 is a Minute gene

Likely

13

M(3)67C

 

   RpS9

67B11

M

 

A deletion removing both RpS17 and RpS9 is Minute

Yes

 

M(3)67C

 

   RpL10Ab

68E1

 

+

 

No

   

   RpS12

69F5

 

+

A deletion that removes RpS12 and RpS4 is Minute

No

   

   RpS4

69F6

  

A deletion that removes RpS12 and RpS4 is Minute

Likely

2

M(3)69E

 

   RpL26

75E4

 

+

 

No

   

   RpLP0

79B2

+

+

 

No

   

   Qm/RpL10

80A

 

M

 

Likely

23

M(3)80

 

   RpL15

80F

 

M

 

Likely

= 11

M(3)80F*

 

Chromosome arm 3R

        

   RpL35A

83A4

  

Lies in a gap in deletion coverage

Likely

3

New?

 

   RpL13A

83B6-7

M

 

Lies in a gap in deletion coverage

Yes

 

M(3)83B*

[52]

   RpL34b

85D15

  

Lies in a gap in deletion coverage

Likely

3

New?

 

   RpS29

85E8

M

M

 

Yes

 

M(3)85E

 

   RpS25

86D8

  

A deletion that removes RpS25 and RpL3 is Minute

Likely

14

M(3)86D

 

   RpL3

86D8

+

  

No

   

   RpS5b

88D6

+

+

 

No

   

   RpL10Aa

88D10

 

+

 

No

   

   RpS20

93A1

  

A deletion that removes RpS20 and RpS30 is Minute

Likely

21

M(3)93A*

 

   RpS30

93A2

  

A deletion that removes RpS20 and RpS30 is Minute

Likely

21

M(3)93A*

 

   RpS3

94E13

M

M

 

Yes

 

M(3)95A

[43]

   RpS19b

95C13

 

+

 

No

   

   RpS27

96C8

 

M

 

Likely

5

M(3)96C

 

   RpL27

96E9-10

 

M

 

Likely

6

M(3)96CF

 

   RpL34a

96F10

 

+

 

No

   

   RpS10a

98A14

 

+

 

No

   

   RpL4

98B6

 

M

 

Likely

6

M(3)98B*

 

   RpS8

99C4

  

Lies in a gap in deletion coverage. 99B aneuploids were Minute [157]

Likely

11

M(3)99B

 

   RpS28a

99D2

  

The Minute phenotype of a deletion removing RpS28a and RpL32 is rescued by a RpL32 transgene [41]

No

   

   RpL32

99D3

  

The Minute phenotype of a deletion removing RpS28a and RpL32 is rescued by a RpL32 transgene [41]

Yes

 

M(3)99D

[41]

   RpS7

99E2

  

Lies in a gap in deletion coverage. 99E-F aneuploids were Minute [157]

Likely

11

M(3)99E

 

   RpL6

100C7

  

Lies in a gap in deletion coverage. 100C-F aneuploids were Minute [157]

Likely

16

M(3)100CF

 

Chromosome 4

        

   RpS3A

101F1

M

M

 

Yes

 

M(4)101

[48]

aBold font indicates the member of a duplicate gene pair that is expressed in a small number of tissues and/or at relatively low levels. bComplete details are given in Additional data file 4. c'M' indicates that mutation or deletion heterozygotes display a Minute bristle phenotype; '+' indicates they are wild type; a blank indicates the absence of appropriate mutations or deletions. dMutations mapped molecularly to a single CRP gene. eDeletions removing several genes, but only a single CRP gene. fJudged according to evidence summarized in previous three columns and presented in detail in Additional data file 4. gThe maximum number of genes that could correspond to the Minute; defined as the number of genes between the relevant deletion breakpoints minus the number of genes with non-Minute mutations. hMinute synonyms from literature sources (see Additional data files 3 and 4). Asterisks indicate new synonyms assigned in this study. iReference demonstrating definite correspondence between a Minute locus and CRP gene. Where no reference is given, the correspondence is shown for the first time in this study.

These 26 cases of proven CRP gene-Minute locus correspondences provide a strong precedent for expecting that other CRP genes are also Minute genes. Although existing reagents do not allow us to demonstrate the correspondences definitively, we judged that a CRP gene very likely corresponds to a genetically defined Minute locus when one or more of the following criteria are fulfilled: a Minute phenotype is seen for a heterozygous multi-gene deletion that uncovers a single CRP gene (for example, M(3)63B/RpL28); a CRP gene lies in a gap in deletion coverage and a molecularly uncharacterized Minute mutation maps to the same region (for example, M(1)8F/RpS28b); or a CRP gene lies in a gap in deletion coverage and previous studies of transient aneuploids document the presence of a Minute locus in the same region (for example, M(3)99E/RpS7). In this way, we identified an additional 36 CRP genes that likely correspond to 34 genetically defined Minute loci (Additional data file 4; summarized in Table 4). Closely linked pairs of CRP genes map to the same regions as M(2)60B and M(3)93A and, as it was impossible to determine whether one or both genes of each pair are haploinsufficient, we have classified all four CRP genes as likely Minute genes. No CRP-like genes mapped to the regions of proven Minute loci. Although five MRP genes map to regions containing Minute loci, it is unlikely that any of them are haploinsufficient: MRP genes are not associated with Minute phenotypes in any other situation, and each of these five MRP genes is closely linked to a CRP gene (Additional data file 4).

We concluded that a further four CRP genes (RpL17, RpL18A, RpL34b and RpL35A) are likely to be Minute genes despite no Minute phenotype having been associated with the genomic region in which they reside. In each of these cases, the CRP gene lies in a gap in deletion coverage (Table 4, Additional data file 4), suggesting that it is a Minute associated with strongly reduced fertility and/or viability, which prevents the establishment of stable deletion stocks (in the absence of a corresponding duplication). Supporting this view, such severe haploinsufficiency also appears to be associated with 15 other CRP genes - all these CRP genes lie in gaps in deletion coverage and they are only considered Minute genes here because they have point or transposon insertion (likely hypomorphic) mutations that cause Minute phenotypes, or because they lie in regions known to harbour Minute loci from the phenotypes of transient aneuploids (Table 4, Additional data file 4).

For all of the 40 CRP genes classified as 'likely Minute genes' (through correlation with genetically proven Minute loci or gaps in deletion coverage), we determined the maximum number of candidate genes that could possibly account for the haploinsufficiency. We used deletions to define the smallest chromosomal interval containing the Minute and then eliminated genes known not to be associated with a Minute phenotype from previous studies or from our own examinations of mutant fly strains. (This task benefited greatly from the recent work of the Bloomington Drosophila Stock Center which, in its efforts to maximize genomic deletion coverage, has systematically generated deletions flanking haploinsufficient loci.) The number of candidate genes defined in this way was always small, ranging from 2 to 33 genes with a median of 8.5 candidate genes per Minute locus (Table 4, Additional data file 4). These data increase our confidence in the likely correspondences between these Minute loci and CRP genes.

The results presented above indicate that 66 CRP genes are, or are likely to be, Minute genes, whereas the remaining 22 CRP genes are not (Table 4 and Additional data files 4 and 5; summarized in Figure 4). CRPs of the large and small ribosomal subunit are encoded by both Minute and non-Minute genes, with no apparent bias. Notably, none of the nine duplicate CRP genes with relatively restricted expression is a Minute, whereas seven of the more highly and widely expressed gene pair members are Minute genes. This is consistent with the idea that only one member of each of these gene pairs contributes significantly to cytoribosomal function in the majority of cells, while the one with restricted expression encodes a component of qualitatively distinct cytoribosomes in certain cell types. As the 'principal' copy of RpS14 or RpL10A is not a Minute, it is unsurprising that the simultaneous heterozygous deletion of both RpS14 genes [95] or both RpL10A genes (in flies with genotypes Df(3L)ED4475/Df(3R)ED10556 or Df(3L)ED4475/Df(3R)ED5660; our observations) does not produce flies with Minute phenotypes. Other possible reasons for different dosage sensitivities among CRP genes are discussed below.
https://static-content.springer.com/image/art%3A10.1186%2Fgb-2007-8-10-r216/MediaObjects/13059_2007_Article_1696_Fig4_HTML.jpg
Figure 4

Summary of Minute locus - CRP gene correspondences. This figure shows the relationship between Minute loci defined by genetic criteria and CRP genes identified using bioinformatics. '=' indicates definite correspondence, '~' indicates probable correspondence. Daggers mark Minute loci that we know or strongly suspect correspond to two CRP genes (as detailed in Table 4 and Additional data file 4).

The one verified Minute locus that does not correspond to a CRP gene is M(1)14C. We mapped this Minute gene to region 14C6 by showing that the deletions Df(1)ED7364 (14A8;14C6) and Df(1)FDD-0230908 (14C6;14E1) are each associated with a Minute phenotype. Moreover, we could rescue these phenotypes, as well as the Minute phenotype associated with the M(1)14C 815-29 point mutation [44], using the small tandem duplication Dp(1;1)FDDP-0024486 (14C4;14D1). The Minute region defined by these experiments contains only two genes: CG4420 and eIF-2α. Significantly, flies heterozygous for P{RS5}eIF-2α 5-HA-1790, an insertion in the 5' untranslated region (UTR) of eIF-2α that creates a likely hypomorphic allele, show a discernable, albeit weak Minute phenotype (our observations). This identifies eIF-2α as M(1)14C. Consistent with this conclusion, flies expressing a dominant-negative eIF-2α protein grow slowly and attain a small body size [96], phenotypes that are typical of the Minute syndrome. eIF-2α is one of the three subunits that constitute eIF2, a key translation initiation factor that delivers the methionine-loaded initiator tRNA to the ribosome by transiently associating with the small cytoribosomal subunit [97]. Although eIF-2α is not a component of cytoribosome complexes isolated by standard biochemical preparations, a reduction in eIF-2α gene dosage might still be expected to adversely affect cytoribosomal function and decrease overall rates of protein synthesis by specifically impairing translation initiation.

Interestingly, the gene encoding eIF-2γ, another subunit of the eIF2 translation initiation factor, is also haploinsufficient. Transcripts from the Su(var)3-9 gene are alternatively spliced to produce two different proteins with distinct functions: one protein is the eIF-2γ translation factor, the other is responsible for suppression of position effect variegation [98]. Mutations that specifically eliminate the suppressor protein are homozygous viable and are not associated with Minute phenotypes [99], but deletions of the entire gene are haplolethal in the absence of P{(ry+), 11. 5kb}, a transgenic construct carrying the complete Su(var)3-9 genomic region (our observations; Additional data file 6). These data indicate that the regions of the Su(var)3-9 transcription unit encoding eIF-2γ are haplolethal. Moreover, it is possible that this haplolethality actually represents an extreme Minute phenotype associated with the eIF-2γ-coding regions; hypomorphic eIF-2γ mutants, if isolated, may show less severe Minute phenotypes.

To assess the possibility that other translation factor genes might also be haploinsufficient/Minute, we examined the heterozygous loss-of-function phenotypes of 68 translation factor genes we identified from BLAST searches and/or Gene Ontology classification (Additional data file 6). We identified no other cases of haploinsufficiency, though five genes could not be assessed with existing deletion and mutation strains. In contrast to the genes encoding the other two subunits of eIF2, the eIF-2β gene is not haploinsufficient.

As mentioned above, we have compared our inventory of Minute genes with the Minute loci defined and named from previous genetic analyses (Additional data file 3). In so doing, we failed to validate the existence of several Minute loci described in the past, namely M(1)3E [55], M(1)4BC [55], M(2)21AB [100102], M(2)44C [55], M(3)76A [55], M(3)82BC [55] and M(3)96A [103]. The existence of some of these loci has been questioned previously and many cases appear to have involved chromosomal aberrations that were unusually complex or point mutations that were mismapped. Our failure to observe a Minute phenotype for deletions of S-adenosylmethionine synthetase (Sam-S), also known as M(2)21AB, is consistent with the phenotypic instability of dominant Sam-S mutations documented previously [100102]. This suggests that mutations in Sam-S can phenocopy Minute mutations under certain conditions, but that Sam-S is not a typical Minute gene.

In summary, CRP genes are likely to correspond to all but one of the 65 Minute loci defined in this study, with the sole exception encoding a translation initiation factor subunit (Figure 4). No MRP or CRP-like genes are unequivocally associated with a Minute phenotype, indicating that the Minute syndrome is specifically related to the function of the cytoribosomes responsible for the majority of cellular protein translation, rather than the function of specialized cytoribosomal variants. Twenty-five percent of CRP genes are not associated with an obvious haploinsufficient phenotype, clearly reinforcing previous findings that not all CRP genes are Minute genes [61, 95, 104].

Discussion

CRP gene haploinsufficiency and the cytoribosome

When one examines the phenotypes of flies carrying chromosomal deletions, one is struck by the remarkable tolerance of Drosophila to aneuploidy: flies heterozygous for deletions of hundreds of kilobases of DNA usually have no obvious dominant phenotypes. For this reason, the haploinsufficiency of single genes is all the more remarkable. It is even more striking that the vast majority of these haploinsufficient genes encode proteins of the cytoribosome and that haploinsufficiency is not apparent for genes encoding components of equally elaborate cellular complexes, such as mitoribosomes or spliceosomes. What accounts for the exquisite dosage sensitivity of CRP genes?

The primary cause of CRP gene haploinsufficiency is reasonably clear: halving the copy number of a CRP gene results in reduced mRNA expression of that CRP [105]. Similarly, depleting CRP mRNAs through antisense- or RNA interference (RNAi)-mediated approaches can also produce Minute phenotypes [106, 107] (SJM and SJL, unpublished data). As there appear to be no compensatory increases in transcription [105], reducing dosages of CRP genes must result in reduced CRP protein levels in the absence of dramatic changes in mRNA stability or CRP protein turnover. How then does the reduction in the level of a single CRP result in impaired cytoribosomal function and reduced general protein synthesis, and how is this manifested as the Minute phenotype?

One possibility, termed the 'balance hypothesis' [108, 109], is linked to the multisubunit nature of the cytoribosome. It posits that an imbalance in the concentrations of CRPs results in the assembly of incomplete and non-functional ribosomal subunits. Indeed, it is known that depletion of individual CRPs in Saccharomyces cerevisiae causes inefficient ribosomal subunit assembly and/or function [110, 111]. Nevertheless, the balance hypothesis also predicts that overexpression of individual CRPs should cause stoichiometric imbalances and phenotypes similar to those produced by underexpression. This prediction is not upheld in either S. cerevisiae [112] or D. melanogaster [113]. Consequently, imbalance per se cannot account for the haploinsufficiency of CRP genes.

A simpler explanation of CRP haploinsufficiency is that a high concentration of cytoribosomes is required for proper cellular functions and that the cytoribosome population decreases sharply when the level of a single CRP is reduced. Cytoribosomes and their components do appear to be required in unusually high quantities: CRP mRNAs are among the most abundant cellular transcripts both in yeast [114] and in flies [60], and can account for 50% of all RNA polymerase II-mediated transcription [89]. What seems critical, however, to the high-level production of fully formed cytoribosomes is that the concentration of each and every CRP never falls below a minimal level. In other words, cytoribosomal assembly is strictly limited by the availability of the least abundant component. This is probably not just a matter of simple self-assembly kinetics as improperly assembled ribosomal subunits and excess CRPs are actively degraded in yeast [111, 115]. Similarly, RNAi-mediated depletion of single CRPs leads to the depletion of other CRPs in flies [64], suggesting the existence of similar degradation processes. Consequently, halving the supply of a single, limiting CRP is expected to halve the number of functional cytoribosomes. This may be tolerated by many cellular processes but will have severe effects wherever high protein synthesis is required, such as bristle formation and oocyte production in flies, or growth of S. cerevisiae in rich media [116]. It appears, therefore, that it is the combination of high demand for cytoribosomes and an assembly mechanism that assures that the level of the least abundant CRP determines the final concentration of cytoribosomes that makes CRP genes so exquisitely and specifically dosage sensitive. This perspective also provides a context for understanding the non-additivity of Minute mutations [117], where combinations of Minute mutations usually do not have a cumulative effect, but rather result in a phenotype similar to that of the most severe individual Minute mutant.

If adequate levels of cytoribosomes depend not so much on precise equimolar CRP concentrations as a minimal concentration of each and every CRP, then we should expect that variation in the expression of different CRPs (above the minimum level) might normally be tolerated in vivo. Such variation could be the result of differences in rates of gene transcription, mRNA translation, or mRNA/protein stability. This view provides a framework for understanding the spectrum of haploinsufficient phenotypes associated with CRP genes, which ranges from no obvious phenotypes, through bristle defects and reduced fecundity and viability, to dominant sterility or haplolethality in the most severe cases. That is, the severity of Minute phenotypes may be related to the rates at which individual CRPs are normally produced [105, 106].

In reality, the explanation of CRP gene haploinsufficiency is probably more complicated than cytoribosome assembly relying simply on minimal CRP concentrations. The exact function, position or stoichiometry of CRPs within the cytoribosome may determine whether its gene is haploinsufficient and the severity of the Minute phenotype. For instance, our finding that the gene encoding the eIF-2α translation initiation factor subunit is a Minute could indicate that haploinsufficient CRP genes encode ribosomal components involved specifically in translation initiation. As another example, RpLP1 and RpLP2 are the only CRPs required in two copies per cytoribosome [15] and, consequently, they must be produced at twice the level of all other CRPs. It is perhaps not surprising, therefore, that both RpLP1 and RpLP2 are haploinsufficient (Table 4) [50]. It may even be the case that the haploinsufficiency of some CRP genes arises by less conventional mechanisms. For example, the introns of 27 CRP genes host genes for small nucleolar RNAs (snoRNAs) [118122], a class of non-coding RNAs that guide post-transcriptional modifications of rRNA molecules necessary for the maturation and incorporation of rRNA into ribosomes [123]. The expression of intronic snoRNAs depends upon the expression and processing of mRNAs from the host gene [124]. Consequently, mutations that reduce expression or splicing of CRP transcripts harboring snoRNAs will simultaneously deplete the cell of both a CRP and properly processed rRNA molecules, thereby impairing cytoribosome biogenesis in two different ways. Although 21 of the 27 CRP genes that carry snoRNA genes within their introns are Minute or likely Minute genes (data not shown), the presence of intronic snoRNA genes cannot be the sole factor determining CRP gene haploinsufficiency. Indeed, we have not found any single factor that clearly determines the degree of dosage sensitivity exhibited by different CRP genes.

Regardless of the exact causes and mechanisms of haploinsufficiency, it is pertinent to ask why the majority of CRPs are expressed so close to the level of sufficiency, such that loss of one gene copy is debilitating, rather than being synthesized in excess? One possibility concerns economics: cytoribosomal synthesis is an incredibly costly affair [89] and excessive CRP production would both be wasteful and monopolize the limited resources of the cell. A second possibility is that CRP levels are normally constrained to guard against inappropriate activation of cell growth, proliferation or apoptosis - processes in which CRPs and cytoribosomes have been postulated to play direct roles [125]. A final and intriguing possibility is that the barely sufficient expression levels of some CRP genes may have evolved as a viral defense mechanism. Cherry et al. [64] found that reducing the levels of 64 of the 79 principal CRPs by RNAi inhibits the propagation of Drosophila C virus in Drosophila adults and cultured cells. Because this virus requires high concentrations of cytoribosomes in its host cell to undergo efficient translation, tightly controlled expression of CRP genes at levels just sufficient for normal growth and development may protect against viral infection and provide a selective advantage. Interestingly, we found a modest correlation between a CRP gene being a Minute and it being able to inhibit virus replication in this assay. Clearly, further work will be required to test whether there is truly a relationship between normal CRP gene expression levels and susceptibility to viruses.

Minute mutations attracted the attention of early geneticists because they were isolated so often in D. melanogaster. In fact, Schultz said in 1929, "...so many have been found that this mutant type is one of the most frequent in Drosophila" [117]. As a considerable number of Minute mutants have also been isolated in other Drosophila species [60], one might be justified in thinking Drosophila are unusually sensitive to CRP gene haploinsufficiency. On the other hand, the phenotypic consequences of CRP gene haploinsufficiency may simply be more noticeable in flies because they include conspicuous changes in external morphology. In fact, 'Minute-ness' may be a widespread phenomenon that is under-recognized because CRP gene haploinsufficiency has different and varied phenotypic consequences in other organisms. Recent research suggests this is the case [116, 126132]. For example, RPS5 haploinsufficiency disrupts cell division and causes developmental and growth phenotypes in Arabidopsis [126]; several CRP genes are haploinsufficient for suppression of nerve sheath tumors in zebrafish [127]; and RPS19 haploinsufficiency is a causative factor of Diamond-Blackfan anemia in humans [128, 130]. In fact, our reliance on the obvious bristle phenotype to distinguish Minute from non-Minute loci may present a biased assessment of CRP gene haploinsufficiency in the fly: it is quite possible that the 22 CRP genes classified as non-Minute in this study are associated with more subtle haploinsufficient phenotypes. How reduced CRP expression gives rise to diverse phenotypes is a mystery that, at least in part, reflects our current ignorance of the regulation and roles of CRPs in different cell types. This is certainly a topic worthy of more research.

Conclusion

We have assessed an idea that has been discussed for more than three decades; namely, that the haploinsufficient Minute loci of Drosophila correspond to the genes encoding protein components of ribosomes [2, 133]. Our results confirm this idea and add important details. We have shown that Minute genes encode proteins of cytoplasmic ribosomes and not mitochondrial ribosomes, and we have defined the subset of CRP genes that are haploinsufficient. While duplicate genes encoding tissue-specific CRPs are not associated with Minute phenotypes, it is not otherwise clear what distinguishes the CRP genes that are haploinsufficient from those that are not. We identified a single Minute gene encoding a different kind of protein, a cytoplasmic translation initiation factor subunit. This hints that haploinsufficient CRP genes may encode proteins specifically involved in translation initiation, although further work is obviously needed to test this idea.

Minute genes account for the vast majority of the haploinsufficient genes in the D. melanogaster genome with effects on fertility and viability strong enough to prevent the recovery of chromosomal deletions in the absence of corresponding duplications. Indeed, there are very few additional genes (for example, dpp [134], Abd-B [135]) or chromosomal regions (for example, Tpl [136], wupA [137], Fs(1)10A [138]) unequivocally associated with haplolethality or haplosterility. (A few other regions have been reported but not investigated in detail.) The most immediate practical use for our data will be in systematic efforts to maximize genome deletion coverage. Knowing which specific genes are haploinsufficient will make it feasible to flank each one as closely as possible with pairs of deletions, or to delete these genes in the presence of duplications or transgenic rescue constructs. Further improvements in deletion coverage will undoubtedly identify and map the remaining haplolethal or haplosterile loci.

Collectively, our inventories of the RP genes and Minute loci of D. melanogaster provide a solid foundation for further studies of RPs, ribosomes, and the causes and consequences of haploinsufficiency in flies and other organisms.

Materials and methods

Bioinformatics

RefSeq human RP sequences were obtained from the National Center for Biotechnology Information [139]. The FlyBase BLASTp service [140] was used to identify high scoring hits from among the annotated proteins of D. melanogaster; tBLASTn was used when orthologs were not identified by a BLASTp search. The ExPASy proteomics server [141] was used to compute the average pI and molecular weight of the RPs. The percentage identity between human and D. melanogaster RP sequences or between D. melanogaster RP pairs was calculated using the NPS@ ClustalW alignment tool at the Pôle Bioinformatique Lyonnais using default parameters [142, 143]. K A /K S values were estimated using the program package PAML [144]. cDNA clone data were obtained from FlyBase [60].

The identification of CRP gene orthologs and the plotting of their evolutionary emergence (Figure 1) were achieved using a combinatorial approach. First, sequences corresponding to the relevant CRP genes from D. melanogaster and Homo sapiens were used as queries in BLASTn, BLASTp, tBLASTn and BLAT searches of the genomes of other Drosophilid and insect species using the FlyBase BLAST server [140] and the UCSC Genome Bioinformatics BLAT server [145, 146]. High scoring matches were judged to be potential orthologs and were analyzed further using the FlyBase OrthoView tool [87]. Second, the coding sequences (CDS) of relevant CRP genes from D. melanogaster and H. sapiens were used as queries in BLASTn searches of the GLEANR CDS prediction sets of other Drosophilid species [140]; phylogenetic trees were then generated from the high scoring matches, with the CDS of S. cerevisisae CRPs as roots, using the ClustalW tools at EMBL-EBI [147]. Third, NCBI Homologene [148] was searched for any relevant homology calls: RpLP0-like, RpL7-like and RpL24-like were found in HomoloGene clusters 102093, 64526 and 9462, respectively. The results of all these analyses were then compared, with the most parsimonious interpretations being used to annotate the dendrogram shown in Figure 1.

Assessing Minute phenotypes and mapping Minutemutations

In order to compile a list of all genetically defined Minute loci, we first catalogued all the Minute loci described in the fly literature [2, 54, 55, 60]. We then inspected deletions for all genomic regions having deletion coverage to confirm or refute the existence of these Minute loci and to identify any new Minute loci that had previously gone undetected. Minute phenotypes were scored primarily by visual inspection of bristle length, although body size, developmental timing, fertility and viability were considered when information was available. Deletion-bearing flies were outcrossed to Oregon-R or Canton-S wild-type strains whenever we could not unambiguously score Minute phenotypes in stocks. The phenotypic effects of deleting or disrupting X-linked genes were assessed only in heterozygous females. Finally, the cytological locations of all verified Minute loci were correlated with the positions of RP genes to identify candidate genes.

To assess RP gene haploinsufficiency directly, we inspected flies heterozygous for deletions and/or mutations of molecularly identified RP genes. Minute phenotypes were scored as described above. For some deletions that had not been characterized molecularly, it was necessary to refine the mapping of breakpoints with complementation tests against molecularly mapped mutations or with polytene chromosome preparations to determine whether RP genes were deleted. (In a few cases, RP genes were classified as lacking deletion coverage when the only existing deletions were not useful in a practical sense owing to their associated chromosomal rearrangements or extremely large size.) A transposable element insertion was judged to disrupt a RP gene if it failed to complement other mutations in the gene, if we saw a Minute phenotype in the insertion strain, or if the transposon is inserted in the protein-coding region or 5' UTR of the gene based on FlyBase annotations [87] or our own BLAST analyses. (By these criteria, many nearby insertions, intronic insertions, and insertions in 3' UTRs were not used in our analysis.) We included molecularly characterized point mutations in our analyses for the few RP genes where they were available. Molecularly uncharacterized Minute point mutations from the Bloomington Stock Center were complementation tested against mutations and deletions known to disrupt or delete specific RP genes.

Fly strains were obtained from the Bloomington, Szeged, Kyoto and Harvard Drosophila Stock Centers. Helene Doerflinger and Daniel St Johnston provided Df(3R)IR16 and Df(3R)MR22 stocks, and Yuri Sedkov and Alexander Mazo provided mRpL16 A , mRpL16 B and mRpL16 C stocks.

Additional data files

The following additional data are available with the online version of this paper. Additional data file 1 is a table comparing the physical characteristics of D. melanogaster and human CRPs, together with their RefSeq accession numbers. Additional data file 2 is a similar table comparing D. melanogaster and human MRPs. Additional data file 3 is a table listing all the Minute loci in a historical context. Additional data file 4 is a table showing our comprehensive genetic analyses of ribosomal protein gene haploinsufficiency. Additional data file 5 is a table listing CRP gene-Minute locus correspondences arranged in alpha-numerical order by RP gene symbol. Additional data file 6 is a table showing our genetic analyses of translation factor gene haploinsufficiency.

Abbreviations

CDS: 

coding sequence

CRP: 

cytoplasmic ribosomal protein

MRP: 

mitochondrial ribosomal protein

RNAi: 

RNA interference

RP: 

ribosomal protein

rRNA: 

ribosomal RNA

snoRNA: 

small nucleolar RNA

UTR: 

untranslated region.

Declarations

Acknowledgements

We would like to thank Bernard Mechler, Erika Viragh, Janos Szidonya, Chris Smith, Barry Honda, Don Sinclair, Kathleen Fitzpatrick, Mirelle Galloni, Lynn Crosby, Helene Doerflinger, Yuri Sedkov, Kathy Matthews and Phil East for data, assistance, helpful discussions or fly strains, and the Bloomington Drosophila Stock Center, the Szeged Drosophila Stock Centre, the Drosophila Genetic Resource Center at the Kyoto Institute of Technology, and the Exelixis Stock Collection at Harvard Medical School for providing the hundreds of strains used in this paper. Special thanks to Rachel Andrade, Kevin Bogart, Stacey Christensen, Jennifer Deal, Megan Deal and Jill Gresens in Bloomington who generated many of the deletions that helped us map haploinsufficient loci with such precision. We also wish to thank Yuk Sang Chan and Terri Morley at the University of Cambridge for help with cytology and fly crosses, and Torill Rolfsen at the University of Oslo for the SEM pictures. SJM and SJL were funded by Cancer Research UK during this project. KRC and TCK were supported by a National Center for Research Resources grant (RR014106) and the Indiana Genomics Initiative. JR and MA were supported by an MRC Programme Grant (G8225539) and a European Union grant (QLRT-1999-30915). GR was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Re911/3-2 and Re911/5-3) and the European Union (QLRI-CT-2000-00915 and LSHG-CT 2000-503433). GM was supported by National Human Genome Research Institute grant HG000739. NK was funded by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology and the Japan Society for the Promotion of Science. PH and ZY were supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.

Authors’ Affiliations

(1)
Growth Regulation Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields
(2)
Department of Genetics, University of Cambridge
(3)
Institute of Genetics, Biologicum, Martin Luther University Halle-Wittenberg
(4)
Institute of Molecular Biosciences, University of Oslo
(5)
Department of Biology, McGill University
(6)
Frontier Science Research Center, University of Miyazaki
(7)
Department of Biology, Indiana University

References

  1. Kay MA, Jacobs-Lorena M: Developmental genetics of ribosome synthesis in Drosophila. Trends Genet. 1987, 3: 347-351. 10.1016/0168-9525(87)90295-2.Google Scholar
  2. Lambertsson A: The Minute genes in Drosophila and their molecular functions. Adv Genet. 1998, 38: 69-134.PubMedGoogle Scholar
  3. Wool IG: The structure and function of eukaryotic ribosomes. Annu Rev Biochem. 1979, 48: 719-754. 10.1146/annurev.bi.48.070179.003443.PubMedGoogle Scholar
  4. Harris EH, Boynton JE, Gillham NW: Chloroplast ribosomes and protein synthesis. Microbiol Rev. 1994, 58: 700-754.PubMedPubMed CentralGoogle Scholar
  5. Wool IG, Chan YL, Gluck A: Structure and evolution of mammalian ribosomal proteins. Biochem Cell Biol. 1995, 73: 933-947.PubMedGoogle Scholar
  6. O'Brien TW: Properties of human mitochondrial ribosomes. IUBMB Life. 2003, 55: 505-513.PubMedGoogle Scholar
  7. Brodersen DE, Nissen P: The social life of ribosomal proteins. FEBS J. 2005, 272: 2098-2108. 10.1111/j.1742-4658.2005.04651.x.PubMedGoogle Scholar
  8. Wool IG: Extraribosomal functions of ribosomal proteins. Trends Biochem Sci. 1996, 21: 164-165. 10.1016/0968-0004(96)20011-8.PubMedGoogle Scholar
  9. Draptchinskaia N, Gustavsson P, Andersson B, Pettersson M, Willig TN, Dianzani I, Ball S, Tchernia G, Klar J, Matsson H, et al: The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia. Nat Genet. 1999, 21: 169-175. 10.1038/5951.PubMedGoogle Scholar
  10. Fisher EM, Beer-Romero P, Brown LG, Ridley A, McNeil JA, Lawrence JB, Willard HF, Bieber FR, Page DC: Homologous ribosomal protein genes on the human X and Y chromosomes: escape from X inactivation and possible implications for Turner syndrome. Cell. 1990, 63: 1205-1218. 10.1016/0092-8674(90)90416-C.PubMedGoogle Scholar
  11. O'Brien TW, O'Brien BJ, Norman RA: Nuclear MRP genes and mitochondrial disease. Gene. 2005, 354: 147-151. 10.1016/j.gene.2005.03.026.PubMedGoogle Scholar
  12. Ruggero D, Pandolfi PP: Does the ribosome translate cancer?. Nat Rev Cancer. 2003, 3: 179-192. 10.1038/nrc1015.PubMedGoogle Scholar
  13. Veuthey AL, Bittar G: Phylogenetic relationships of fungi, plantae, and animalia inferred from homologous comparison of ribosomal proteins. J Mol Evol. 1998, 47: 81-92. 10.1007/PL00006365.PubMedGoogle Scholar
  14. Nakao A, Yoshihama M, Kenmochi N: RPG: the Ribosomal Protein Gene database. Nucleic Acids Res. 2004, 32: D168-170. 10.1093/nar/gkh004.PubMedPubMed CentralGoogle Scholar
  15. Wahl MC, Moller W: Structure and function of the acidic ribosomal stalk proteins. Curr Protein Pept Sci. 2002, 3: 93-106. 10.2174/1389203023380756.PubMedGoogle Scholar
  16. Koc EC, Burkhart W, Blackburn K, Moseley A, Spremulli LL: The small subunit of the mammalian mitochondrial ribosome. Identification of the full complement of ribosomal proteins present. J Biol Chem. 2001, 276: 19363-19374. 10.1074/jbc.M106510200.Google Scholar
  17. Koc EC, Burkhart W, Blackburn K, Moyer MB, Schlatzer DM, Moseley A, Spremulli LL: The large subunit of the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present. J Biol Chem. 2001, 276: 43958-43969. 10.1074/jbc.M106510200.PubMedGoogle Scholar
  18. HUGO Gene Nomenclature Committee. [http://www.genenames.org/]
  19. Lambertsson AG, Rasmuson SB, Bloom GD: The ribosomal proteins of Drosophila melanogaster. I. Characterization in polyacrylamide gel of proteins from larval, adult, and ammonium chloride-treated ribosomes. Mol Gen Genet. 1970, 108: 349-357. 10.1007/BF00267772.PubMedGoogle Scholar
  20. Lambertsson AG: The ribosomal proteins of Drosophila melanogaster. II. Comparison of protein patterns of ribosomes from larvae, pupae and adult flies by two-dimensional polyacrylamide gel electrophoresis. Mol Gen Genet. 1972, 118: 215-222. 10.1007/BF00333458.PubMedGoogle Scholar
  21. Berger E: The ribosomes of Drosophila. I. Subunit and protein composition. Mol Gen Genet. 1974, 128: 1-9. 10.1007/BF00267290.PubMedGoogle Scholar
  22. Berger EM, Weber L: The ribosomes of Drosophila. II. Studies on intraspecific variation. Genetics. 1974, 78: 1173-1183.PubMedPubMed CentralGoogle Scholar
  23. Lambertsson AG: The ribosomal proteins of Drosophila melanogaster. 3. Further studies on the ribosomal protein composition during development. Mol Gen Genet. 1974, 128: 241-247. 10.1007/BF00267113.PubMedGoogle Scholar
  24. Lambertsson AG: The ribosomal proteins of Drosophila melanogaster. IV. Characterization by two-dimensional gel electrophoresis of the ribosomal proteins from nine postembryonic developmental stages. Mol Gen Genet. 1975, 139: 133-144.PubMedGoogle Scholar
  25. Vaslet C, Berger E: The ribosomes of Drosophila. IV. Electrophoretic identify among ribosomal subunit proteins from wild type and mutant D. melanogaster and D. simulans. Mol Gen Genet. 1976, 147: 189-194. 10.1007/BF00267570.PubMedGoogle Scholar
  26. Weber L, Berger E, Vaslet C, Yedvobnick B: The ribosomes of Drosophila. III. RNA and protein homology between D. melanogaster and D. virilis. Genetics. 1976, 84: 573-585.PubMedPubMed CentralGoogle Scholar
  27. Berger E: The ribosomes of Drosophila. Normal and defective ribosome biosynthesis in Drosophila cell cultures. Mol Gen Genet. 1977, 155: 35-40. 10.1007/BF00268558.PubMedGoogle Scholar
  28. Fekete E, Lambertsson A: Imaginal disc ribosomal proteins of D. melanogaster. Mol Gen Genet. 1978, 159: 85-87. 10.1007/BF00401751.Google Scholar
  29. Chooi WY, Sabatini LM, Macklin M, Fraser D: Group fractionation and determination of the number of ribosomal subunit proteins from Drosophila melanogaster embryos. Biochemistry. 1980, 19: 1425-1433. 10.1021/bi00548a025.PubMedGoogle Scholar
  30. Chooi WY: A comparison of the ribosomal proteins of Drosophila ovary, adult, and embryo. Mol Gen Genet. 1981, 184: 342-346. 10.1007/BF00352502.PubMedGoogle Scholar
  31. Kay MA, Jacobs-Lorena M: Selective translational regulation of ribosomal protein gene expression during early development of Drosophila melanogaster. Mol Cell Biol. 1985, 5: 3583-3592.PubMedPubMed CentralGoogle Scholar
  32. Chooi WY: Purification of Drosophila ribosomal proteins. Isolation of proteins S8, S13, S14, S16, S19, S20/L24, S22/L26, S24, S25/S27, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 7/8, 9, and 11. Biochemistry. 1980, 19: 3469-3476. 10.1021/bi00556a010.PubMedGoogle Scholar
  33. Chooi WY, Macklin MD, Leiby KR, Hong TH, Scofield SR, Sabatini LM, Burns DK: Purification of Drosophila acidic ribosomal proteins. Eur J Biochem. 1982, 127: 199-205. 10.1111/j.1432-1033.1982.tb06856.x.PubMedGoogle Scholar
  34. Alonso J, Santaren JF: Characterization of the Drosophila melanogaster ribosomal proteome. J Proteome Res. 2006, 5: 2025-2032. 10.1021/pr0601483.PubMedGoogle Scholar
  35. Planta RJ, Mager WH: The list of cytoplasmic ribosomal proteins of Saccharomyces cerevisiae. Yeast. 1998, 14: 471-477. 10.1002/(SICI)1097-0061(19980330)14:5<471::AID-YEA241>3.0.CO;2-U.PubMedGoogle Scholar
  36. Barakat A, Szick-Miranda K, Chang IF, Guyot R, Blanc G, Cooke R, Delseny M, Bailey-Serres J: The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome. Plant Physiol. 2001, 127: 398-415. 10.1104/pp.127.2.398.PubMedPubMed CentralGoogle Scholar
  37. Kenmochi N, Kawaguchi T, Rozen S, Davis E, Goodman N, Hudson TJ, Tanaka T, Page DC: A map of 75 human ribosomal protein genes. Genome Res. 1998, 8: 509-523.PubMedGoogle Scholar
  38. Kenmochi N, Suzuki T, Uechi T, Magoori M, Kuniba M, Higa S, Watanabe K, Tanaka T: The human mitochondrial ribosomal protein genes: mapping of 54 genes to the chromosomes and implications for human disorders. Genomics. 2001, 77: 65-70. 10.1006/geno.2001.6622.PubMedGoogle Scholar
  39. Uechi T, Tanaka T, Kenmochi N: A complete map of the human ribosomal protein genes: assignment of 80 genes to the cytogenetic map and implications for human disorders. Genomics. 2001, 72: 223-230. 10.1006/geno.2000.6470.PubMedGoogle Scholar
  40. Yoshihama M, Uechi T, Asakawa S, Kawasaki K, Kato S, Higa S, Maeda N, Minoshima S, Tanaka T, Shimizu N, Kenmochi N: The human ribosomal protein genes: sequencing and comparative analysis of 73 genes. Genome Res. 2002, 12: 379-390. 10.1101/gr.214202.PubMedPubMed CentralGoogle Scholar
  41. Kongsuwan K, Yu Q, Vincent A, Frisardi MC, Rosbash M, Lengyel JA, Merriam J: A Drosophila Minute gene encodes a ribosomal protein. Nature. 1985, 317: 555-558. 10.1038/317555a0.PubMedGoogle Scholar
  42. Hart K, Klein T, Wilcox M: A Minute encoding a ribosomal protein enhances wing morphogenesis mutants. Mech Dev. 1993, 43: 101-110. 10.1016/0925-4773(93)90028-V.PubMedGoogle Scholar
  43. Andersson S, Saeboe-Larssen S, Lambertsson A, Merriam J, Jacobs-Lorena M: A Drosophila third chromosome Minute locus encodes a ribosomal protein. Genetics. 1994, 137: 513-520.PubMedPubMed CentralGoogle Scholar
  44. McKim KS, Dahmus JB, Hawley RS: Cloning of the Drosophila melanogaster meiotic recombination gene mei-218: a genetic and molecular analysis of interval 15E. Genetics. 1996, 144: 215-228.PubMedPubMed CentralGoogle Scholar
  45. Saeboe-Larssen S, Lambertsson A: A novel Drosophila Minute locus encodes ribosomal protein S13. Genetics. 1996, 143: 877-885.PubMedGoogle Scholar
  46. Schmidt A, Hollmann M, Schafer U: A newly identified Minute locus, M(2)32D, encodes the ribosomal protein L9 in Drosophila melanogaster. Mol Gen Genet. 1996, 251: 381-387.PubMedGoogle Scholar
  47. Saeboe-Larssen S, Urbanczyk Mohebi B, Lambertsson A: The Drosophila ribosomal protein L14-encoding gene, identified by a novel Minute mutation in a dense cluster of previously undescribed genes in cytogenetic region 66D. Mol Gen Genet. 1997, 255: 141-151. 10.1007/s004380050482.PubMedGoogle Scholar
  48. van Beest M, Mortin M, Clevers H: Drosophila RpS3a, a novel Minute gene situated between the segment polarity genes cubitus interruptus and dTCF. Nucleic Acids Res. 1998, 26: 4471-4475. 10.1093/nar/26.19.4471.PubMedPubMed CentralGoogle Scholar
  49. Torok I, Herrmann-Horle D, Kiss I, Tick G, Speer G, Schmitt R, Mechler BM: Down-regulation of RpS21, a putative translation initiation factor interacting with P40, produces viable Minute imagos and larval lethality with overgrown hematopoietic organs and imaginal discs. Mol Cell Biol. 1999, 19: 2308-2321.PubMedPubMed CentralGoogle Scholar
  50. Fauvarque MO, Laurenti P, Boivin A, Bloyer S, Griffin-Shea R, Bourbon HM, Dura JM: Dominant modifiers of the polyhomeotic extra-sex-combs phenotype induced by marked P element insertional mutagenesis in Drosophila. Genet Res. 2001, 78: 137-148.PubMedGoogle Scholar
  51. Marygold SJ, Coelho CM, Leevers SJ: Genetic analysis of RpL38 and RpL5, two Minute genes located in the centric heterochromatin of chromosome 2 of Drosophila melanogaster. Genetics. 2005, 169: 683-695. 10.1534/genetics.104.034124.PubMedPubMed CentralGoogle Scholar
  52. Alexander SJ, Woodling NS, Yedvobnick B: Insertional inactivation of the L13a ribosomal protein gene of Drosophila melanogaster identifies a new Minute locus. Gene. 2006, 368: 46-52. 10.1016/j.gene.2005.10.005.PubMedGoogle Scholar
  53. Tyler DM, Li W, Zhuo N, Pellock B, Baker NE: Genes affecting cell competition in Drosophila. Genetics. 2007, 175: 643-657. 10.1534/genetics.106.061929.PubMedPubMed CentralGoogle Scholar
  54. Lindsley DL, Sandler L, Baker BS, Carpenter AT, Denell RE, Hall JC, Jacobs PA, Miklos GL, Davis BK, Gethmann RC, et al: Segmental aneuploidy and the genetic gross structure of the Drosophila genome. Genetics. 1972, 71: 157-184.PubMedPubMed CentralGoogle Scholar
  55. Lindsley DL, Zimm GG: The Genome of Drosophila melanogaster. 1992, San Diego, California: Academic PressGoogle Scholar
  56. Stewart B, Merriam JR: Segmental aneuploidy of the X-chromosome. Drosophila Information Service. 1973, 50: 167-170.Google Scholar
  57. The Bloomington Drosophila Stock Center: General Information About the Bloomington Deficiency Kit. [http://flystocks.bio.indiana.edu/Browse/df-dp/dfkit-info.htm]
  58. McConkey EH, Bielka H, Gordon J, Lastick SM, Lin A, Ogata K, Reboud JP, Traugh JA, Traut RR, Warner JR, et al: Proposed uniform nomenclature for mammalian ribosomal proteins. Mol Gen Genet. 1979, 169: 1-6. 10.1007/BF00267538.PubMedGoogle Scholar
  59. Wool IG, Chan YL, Gluck A, Suzuki K: The primary structure of rat ribosomal proteins P0, P1, and P2 and a proposal for a uniform nomenclature for mammalian and yeast ribosomal proteins. Biochimie. 1991, 73: 861-870. 10.1016/0300-9084(91)90127-M.PubMedGoogle Scholar
  60. FlyBase: A Database of Drosophila Genes and Genomes. [http://flybase.org/]
  61. Cramton SE, Laski FA: string of pearls encodes Drosophila ribosomal protein S2, has Minute-like characteristics, and is required during oogenesis. Genetics. 1994, 137: 1039-1048.PubMedPubMed CentralGoogle Scholar
  62. Galloni M, Edgar BA: Cell-autonomous and non-autonomous growth-defective mutants of Drosophila melanogaster. Development. 1999, 126: 2365-2375.PubMedGoogle Scholar
  63. Galloni M: Bonsai, a ribosomal protein S15 homolog, involved in gut mitochondrial activity and systemic growth. Dev Biol. 2003, 264: 482-494. 10.1016/j.ydbio.2003.08.021.PubMedGoogle Scholar
  64. Cherry S, Doukas T, Armknecht S, Whelan S, Wang H, Sarnow P, Perrimon N: Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition. Genes Dev. 2005, 19: 445-452. 10.1101/gad.1267905.PubMedPubMed CentralGoogle Scholar
  65. Koyama Y, Katagiri S, Hanai S, Uchida K, Miwa M: Poly(ADP-ribose) polymerase interacts with novel Drosophila ribosomal proteins, L22 and L23a, with unique histone-like amino-terminal extensions. Gene. 1999, 226: 339-345. 10.1016/S0378-1119(98)00529-0.PubMedGoogle Scholar
  66. Lee HS, Simon JA, Lis JT: Structure and expression of ubiquitin genes of Drosophila melanogaster. Mol Cell Biol. 1988, 8: 4727-4735.PubMedPubMed CentralGoogle Scholar
  67. Cabrera y, Poch HL, Arribas C, Izquierdo M: Sequence of a Drosophila cDNA encoding a ubiquitin gene fusion to a 52-aa ribosomal protein tail. Nucleic Acids Res. 1990, 18: 3994-10.1093/nar/18.13.3994.Google Scholar
  68. Cabrera HL, Barrio R, Arribas C: Structure and expression of the Drosophila ubiquitin-52-amino-acid fusion-protein gene. Biochem J. 1992, 286: 281-288.PubMedPubMed CentralGoogle Scholar
  69. Barrio R, del Arco A, Cabrera HL, Arribas C: Structure and expression of the Drosophila ubiquitin-80-amino-acid fusion-protein gene. Biochem J. 1994, 302: 237-244.PubMedPubMed CentralGoogle Scholar
  70. Vaslet CA, O'Connell P, Izquierdo M, Rosbash M: Isolation and mapping of a cloned ribosomal protein gene of Drosophila melanogaster. Nature. 1980, 285: 674-676. 10.1038/285674a0.PubMedGoogle Scholar
  71. Burns DK, Stark BC, Macklin MD, Chooi WY: Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster. Mol Cell Biol. 1984, 4: 2643-2652.PubMedPubMed CentralGoogle Scholar
  72. Qian S, Zhang JY, Kay MA, Jacobs-Lorena M: Structural analysis of the Drosophila rpA1 gene, a member of the eucaryotic 'A' type ribosomal protein family. Nucleic Acids Res. 1987, 15: 987-1003. 10.1093/nar/15.3.987.PubMedPubMed CentralGoogle Scholar
  73. Kay MA, Zhang JY, Jacobs-Lorena M: Identification and germline transformation of the ribosomal protein rp21 gene of Drosophila: complementation analysis with the Minute QIII locus reveals nonidentity. Mol Gen Genet. 1988, 213: 354-358. 10.1007/BF00339602.PubMedGoogle Scholar
  74. Uechi T, Maeda N, Tanaka T, Kenmochi N: Functional second genes generated by retrotransposition of the X-linked ribosomal protein genes. Nucleic Acids Res. 2002, 30: 5369-5375. 10.1093/nar/gkf696.PubMedPubMed CentralGoogle Scholar
  75. Zhang Z, Harrison P, Gerstein M: Identification and analysis of over 2000 ribosomal protein pseudogenes in the human genome. Genome Res. 2002, 12: 1466-1482. 10.1101/gr.331902.PubMedPubMed CentralGoogle Scholar
  76. Brown SJ, Rhoads DD, Stewart MJ, Van Slyke B, Chen IT, Johnson TK, Denell RE, Roufa DJ: Ribosomal protein S14 is encoded by a pair of highly conserved, adjacent genes on the X chromosome of Drosophila melanogaster. Mol Cell Biol. 1988, 8: 4314-4321.PubMedPubMed CentralGoogle Scholar
  77. Harrison PM, Milburn D, Zhang Z, Bertone P, Gerstein M: Identification of pseudogenes in the Drosophila melanogaster genome. Nucleic Acids Res. 2003, 31: 1033-1037. 10.1093/nar/gkg169.PubMedPubMed CentralGoogle Scholar
  78. Parisi M, Nuttall R, Naiman D, Bouffard G, Malley J, Andrews J, Eastman S, Oliver B: Paucity of genes on the Drosophila X chromosome showing male-biased expression. Science. 2003, 299: 697-700. 10.1126/science.1079190.PubMedPubMed CentralGoogle Scholar
  79. Betran E, Thornton K, Long M: Retroposed new genes out of the X in Drosophila. Genome Res. 2002, 12: 1854-1859. 10.1101/gr.6049.PubMedPubMed CentralGoogle Scholar
  80. Lifschytz E, Lindsley DL: The role of X-chromosome inactivation during spermatogenesis. Proc Natl Acad Sci USA. 1972, 69: 182-186. 10.1073/pnas.69.1.182.PubMedPubMed CentralGoogle Scholar
  81. Shigenobu S, Kitadate Y, Noda C, Kobayashi S: Molecular characterization of embryonic gonads by gene expression profiling in Drosophila melanogaster. Proc Natl Acad Sci USA. 2006, 103: 13728-13733. 10.1073/pnas.0603767103.PubMedPubMed CentralGoogle Scholar
  82. Kai T, Williams D, Spradling AC: The expression profile of purified Drosophila germline stem cells. Dev Biol. 2005, 283: 486-502. 10.1016/j.ydbio.2005.04.018.PubMedGoogle Scholar
  83. Saveanu C, Bienvenu D, Namane A, Gleizes PE, Gas N, Jacquier A, Fromont-Racine M: Nog2p, a putative GTPase associated with pre-60S subunits and required for late 60S maturation steps. EMBO J. 2001, 20: 6475-6484. 10.1093/emboj/20.22.6475.PubMedPubMed CentralGoogle Scholar
  84. Zhao W, Bidwai AP, Glover CV: Interaction of casein kinase II with ribosomal protein L22 of Drosophila melanogaster. Biochem Biophys Res Commun. 2002, 298: 60-66. 10.1016/S0006-291X(02)02396-3.PubMedGoogle Scholar
  85. Shigenobu S, Arita K, Kitadate Y, Noda C, Kobayashi S: Isolation of germline cells from Drosophila embryos by flow cytometry. Dev Growth Differentiation. 2006, 48: 49-57. 10.1111/j.1440-169X.2006.00845.x.Google Scholar
  86. Stewart MJ, Denell R: The Drosophila ribosomal protein S6 gene includes a 3' triplication that arose by unequal crossing-over. Mol Biol Evol. 1993, 10: 1041-1047.PubMedGoogle Scholar
  87. FlyBase: GBrowse - D. melanogaster. [http://flybase.org/cgi-bin/gbrowse/dmel/]
  88. Elgin SC, Grewal SI: Heterochromatin: silence is golden. Curr Biol. 2003, 13: R895-898. 10.1016/j.cub.2003.11.006.PubMedGoogle Scholar
  89. Warner JR: The economics of ribosome biosynthesis in yeast. Trends Biochem Sci. 1999, 24: 437-440. 10.1016/S0968-0004(99)01460-7.PubMedGoogle Scholar
  90. Ryder E, Blows F, Ashburner M, Bautista-Llacer R, Coulson D, Drummond J, Webster J, Gubb D, Gunton N, Johnson G, et al: The DrosDel collection: a set of P-element insertions for generating custom chromosomal aberrations in Drosophila melanogaster. Genetics. 2004, 167: 797-813. 10.1534/genetics.104.026658.PubMedPubMed CentralGoogle Scholar
  91. Ryder E, Ashburner M, Bautista-Llacer R, Drummond J, Webster J, Johnson G, Morley T, Chan YS, Blows F, Coulson D, et al: The DrosDel Deletion Collection: A Drosophila Genomewide Chromosomal Deficiency Resource. Genetics. 2007, 177: 615-629. 10.1534/genetics.107.076216.PubMedPubMed CentralGoogle Scholar
  92. Parks AL, Cook KR, Belvin M, Dompe NA, Fawcett R, Huppert K, Tan LR, Winter CG, Bogart KP, Deal JE, et al: Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nat Genet. 2004, 36: 288-292. 10.1038/ng1312.PubMedGoogle Scholar
  93. Bellen HJ, Levis RW, Liao G, He Y, Carlson JW, Tsang G, Evans-Holm M, Hiesinger PR, Schulze KL, Rubin GM, et al: The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics. 2004, 167: 761-781. 10.1534/genetics.104.026427.PubMedPubMed CentralGoogle Scholar
  94. Thibault ST, Singer MA, Miyazaki WY, Milash B, Dompe NA, Singh CM, Buchholz R, Demsky M, Fawcett R, Francis-Lang HL, et al: A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat Genet. 2004, 36: 283-287. 10.1038/ng1314.PubMedGoogle Scholar
  95. Dorer DR, Anane-Firempong A, Christensen AC: Ribosomal protein S14 is not responsible for the Minute phenotype associated with the M(1)7C locus in Drosophila melanogaster. Mol Gen Genet. 1991, 230: 8-11. 10.1007/BF00290642.PubMedGoogle Scholar
  96. Qu S, Perlaky SE, Organ EL, Crawford D, Cavener DR: Mutations at the Ser50 residue of translation factor eIF-2alpha dominantly affect developmental rate, body weight, and viability of Drosophila melanogaster. Gene Expression. 1997, 6: 349-360.PubMedGoogle Scholar
  97. Kimball SR: Eukaryotic initiation factor eIF2. Int J Biochem Cell Biol. 1999, 31: 25-29. 10.1016/S1357-2725(98)00128-9.PubMedGoogle Scholar
  98. Krauss V, Reuter G: Two genes become one: the genes encoding heterochromatin protein Su(var)3-9 and translation initiation factor subunit eIF-2gamma are joined to a dicistronic unit in holometabolic insects. Genetics. 2000, 156: 1157-1167.PubMedPubMed CentralGoogle Scholar
  99. Schotta G, Ebert A, Krauss V, Fischer A, Hoffmann J, Rea S, Jenuwein T, Dorn R, Reuter G: Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing. EMBO J. 2002, 21: 1121-1131. 10.1093/emboj/21.5.1121.PubMedPubMed CentralGoogle Scholar
  100. Persson K: A Minute mutant with suppressor effect on the eye-colour gene zeste in Drosophila melanogaster. Hereditas. 1976, 82: 57-62.PubMedGoogle Scholar
  101. Larsson J, Zhang J, Rasmuson-Lestander A: Mutations in the Drosophila melanogaster gene encoding S-adenosylmethionine synthetase suppress position-effect variegation. Genetics. 1996, 143: 887-896.PubMedPubMed CentralGoogle Scholar
  102. Larsson J, Rasmuson-Lestander A: Somatic and germline clone analysis in mutants of the S-adenosylmethionine synthetase encoding gene in Drosophila melanogaster. FEBS Lett. 1998, 427: 119-123. 10.1016/S0014-5793(98)00408-6.PubMedGoogle Scholar
  103. Gonzalez C, Molina I, Casal J, Ripoll P: Gross genetic dissection and interaction of the chromosomal region 95E;96F of Drosophila melanogaster. Genetics. 1989, 123: 371-377.PubMedPubMed CentralGoogle Scholar
  104. Frolov MV, Birchler JA: Mutation in P0, a dual function ribosomal protein/apurinic/apyrimidinic endonuclease, modifies gene expression and position effect variegation in Drosophila. Genetics. 1998, 150: 1487-1495.PubMedPubMed CentralGoogle Scholar
  105. Saeboe-Larssen S, Lyamouri M, Merriam J, Oksvold MP, Lambertsson A: Ribosomal protein insufficiency and the Minute syndrome in Drosophila: a dose-response relationship. Genetics. 1998, 148: 1215-1224.PubMedPubMed CentralGoogle Scholar
  106. Patel R, Jacobs-Lorena M: Generation of Minute phenotypes by a transformed antisense ribosomal protein gene. Dev Genet. 1992, 13: 256-263. 10.1002/dvg.1020130403.PubMedGoogle Scholar
  107. Enerly E, Larsson J, Lambertsson A: Silencing the Drosophila ribosomal protein L14 gene using targeted RNA interference causes distinct somatic anomalies. Gene. 2003, 320: 41-48. 10.1016/S0378-1119(03)00827-8.PubMedGoogle Scholar
  108. Veitia RA: Exploring the etiology of haploinsufficiency. Bioessays. 2002, 24: 175-184. 10.1002/bies.10023.PubMedGoogle Scholar
  109. Papp B, Pal C, Hurst LD: Dosage sensitivity and the evolution of gene families in yeast. Nature. 2003, 424: 194-197. 10.1038/nature01771.PubMedGoogle Scholar
  110. Baronas-Lowell DM, Warner JR: Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1990, 10: 5235-5243.PubMedPubMed CentralGoogle Scholar
  111. Zhao Y, Sohn JH, Warner JR: Autoregulation in the biosynthesis of ribosomes. Mol Cell Biol. 2003, 23: 699-707. 10.1128/MCB.23.2.699-707.2003.PubMedPubMed CentralGoogle Scholar
  112. Warner JR, Mitra G, Schwindinger WF, Studeny M, Fried HM: Saccharomyces cerevisiae coordinates accumulation of yeast ribosomal proteins by modulating mRNA splicing, translational initiation, and protein turnover. Mol Cell Biol. 1985, 5: 1512-1521.PubMedPubMed CentralGoogle Scholar
  113. Tamate HB, Patel RC, Riedl AE, Jacobs-Lorena M: Overproduction and translational regulation of rp49 ribosomal protein mRNA in transgenic Drosophila carrying extra copies of the gene. Mol Gen Genet. 1990, 221: 171-175. 10.1007/BF00261717.PubMedGoogle Scholar
  114. Velculescu VE, Zhang L, Zhou W, Vogelstein J, Basrai MA, Bassett DE, Hieter P, Vogelstein B, Kinzler KW: Characterization of the yeast transcriptome. Cell. 1997, 88: 243-251. 10.1016/S0092-8674(00)81845-0.PubMedGoogle Scholar
  115. Tsay YF, Thompson JR, Rotenberg MO, Larkin JC, Woolford JL: Ribosomal protein synthesis is not regulated at the translational level in Saccharomyces cerevisiae: balanced accumulation of ribosomal proteins L16 and rp59 is mediated by turnover of excess protein. Genes Dev. 1988, 2: 664-676. 10.1101/gad.2.6.664.PubMedGoogle Scholar
  116. Deutschbauer AM, Jaramillo DF, Proctor M, Kumm J, Hillenmeyer ME, Davis RW, Nislow C, Giaever G: Mechanisms of haploinsufficiency revealed by genome-wide profiling in yeast. Genetics. 2005, 169: 1915-1925. 10.1534/genetics.104.036871.PubMedPubMed CentralGoogle Scholar
  117. Schultz J: The Minute reaction in the development of Drosophila melanogaster. Genetics. 1929, 14: 366-419.PubMedPubMed CentralGoogle Scholar
  118. Lyamouri M, Enerly E, Kress H, Lambertsson A: Conservation of gene order, structure and sequence between three closely linked genes in Drosophila melanogaster and Drosophila virilis. Gene. 2002, 282: 199-206. 10.1016/S0378-1119(01)00831-9.PubMedGoogle Scholar
  119. Yuan G, Klambt C, Bachellerie JP, Brosius J, Huttenhofer A: RNomics in Drosophila melanogaster: identification of 66 candidates for novel non-messenger RNAs. Nucleic Acids Res. 2003, 31: 2495-2507. 10.1093/nar/gkg361.PubMedPubMed CentralGoogle Scholar
  120. Accardo MC, Giordano E, Riccardo S, Digilio FA, Iazzetti G, Calogero RA, Furia M: A computational search for box C/D snoRNA genes in the Drosophila melanogaster genome. Bioinformatics. 2004, 20: 3293-3301. 10.1093/bioinformatics/bth394.PubMedGoogle Scholar
  121. Huang ZP, Zhou H, Liang D, Qu LH: Different expression strategy: multiple intronic gene clusters of box H/ACA snoRNA in Drosophila melanogaster. J Mol Biol. 2004, 341: 669-683. 10.1016/j.jmb.2004.06.041.PubMedGoogle Scholar
  122. Huang ZP, Zhou H, He HL, Chen CL, Liang D, Qu LH: Genome-wide analyses of two families of snoRNA genes from Drosophila melanogaster, demonstrating the extensive utilization of introns for coding of snoRNAs. Rna. 2005, 11: 1303-1316. 10.1261/rna.2380905.PubMedPubMed CentralGoogle Scholar
  123. Kiss T: Small nucleolar RNAs: an abundant group of noncoding RNAs with diverse cellular functions. Cell. 2002, 109: 145-148. 10.1016/S0092-8674(02)00718-3.PubMedGoogle Scholar
  124. Tycowski KT, Steitz JA: Non-coding snoRNA host genes in Drosophila: expression strategies for modification guide snoRNAs. Eur J Cell Biol. 2001, 80: 119-125. 10.1078/0171-9335-00150.PubMedGoogle Scholar
  125. Rudra D, Warner JR: What better measure than ribosome synthesis?. Genes Dev. 2004, 18: 2431-2436. 10.1101/gad.1256704.PubMedGoogle Scholar
  126. Weijers D, Franke-van Dijk M, Vencken RJ, Quint A, Hooykaas P, Offringa R: An Arabidopsis Minute-like phenotype caused by a semi-dominant mutation in a RIBOSOMAL PROTEIN S5 gene. Development. 2001, 128: 4289-4299.PubMedGoogle Scholar
  127. Amsterdam A, Sadler KC, Lai K, Farrington S, Bronson RT, Lees JA, Hopkins N: Many ribosomal protein genes are cancer genes in zebrafish. PLoS Biol. 2004, 2: E139-10.1371/journal.pbio.0020139.PubMedPubMed CentralGoogle Scholar
  128. Gazda HT, Zhong R, Long L, Niewiadomska E, Lipton JM, Ploszynska A, Zaucha JM, Vlachos A, Atsidaftos E, Viskochil DH, et al: RNA and protein evidence for haplo-insufficiency in Diamond-Blackfan anaemia patients with RPS19 mutations. Br J Haematol. 2004, 127: 105-113. 10.1111/j.1365-2141.2004.05152.x.PubMedGoogle Scholar
  129. Oliver ER, Saunders TL, Tarle SA, Glaser T: Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse Minute. Development. 2004, 131: 3907-3920. 10.1242/dev.01268.PubMedPubMed CentralGoogle Scholar
  130. Leger-Silvestre I, Caffrey JM, Dawaliby R, Alvarez-Arias DA, Gas N, Bertolone SJ, Gleizes PE, Ellis SR: Specific role for yeast homologs of the Diamond Blackfan anemia-associated Rps19 protein in ribosome synthesis. J Biol Chem. 2005, 280: 38177-38185. 10.1074/jbc.M506916200.PubMedGoogle Scholar
  131. Panic L, Tamarut S, Sticker-Jantscheff M, Barkic M, Solter D, Uzelac M, Grabusic K, Volarevic S: Ribosomal protein S6 gene haploinsufficiency is associated with activation of a p53-dependent checkpoint during gastrulation. Mol Cell Biol. 2006, 26: 8880-8891. 10.1128/MCB.00751-06.PubMedPubMed CentralGoogle Scholar
  132. Panic L, Montagne J, Cokaric M, Volarevic S: S6-haploinsufficiency activates the p53 tumor suppressor. Cell Cycle. 2007, 6: 20-24.PubMedGoogle Scholar
  133. Huang SL, Baker BS: The mutability of the Minute loci of Drosophila melanogaster with ethyl methanesulfonate. Mutat Res. 1976, 34: 407-414.PubMedGoogle Scholar
  134. Spencer FA, Hoffmann FM, Gelbart WM: Decapentaplegic: a gene complex affecting morphogenesis in Drosophila melanogaster. Cell. 1982, 28: 451-461. 10.1016/0092-8674(82)90199-4.PubMedGoogle Scholar
  135. Karch F, Weiffenbach B, Peifer M, Bender W, Duncan I, Celniker S, Crosby M, Lewis EB: The abdominal region of the bithorax complex. Cell. 1985, 43: 81-96. 10.1016/0092-8674(85)90014-5.PubMedGoogle Scholar
  136. Dorer DR, Rudnick JA, Moriyama EN, Christensen AC: A family of genes clustered at the Triplo-lethal locus of Drosophila melanogaster has an unusual evolutionary history and significant synteny with Anopheles gambiae. Genetics. 2003, 165: 613-621.PubMedPubMed CentralGoogle Scholar
  137. Prado A, Canal I, Ferrus A: The haplolethal region at the 16F gene cluster of Drosophila melanogaster: structure and function. Genetics. 1999, 151: 163-175.PubMedPubMed CentralGoogle Scholar
  138. Lefevre G: The eccentricity of vermilion deficiencies in Drosophila melanogaster. Genetics. 1969, 63: 589-600.PubMedPubMed CentralGoogle Scholar
  139. National Center for Biotechnology Information: Entrez Protein. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=Protein]
  140. FlyBase: BLAST. [http://flybase.org/blast/]
  141. ExPASy Proteomics Server: Compute pI/Mw tool. [http://www.expasy.org/tools/pi_tool.html]
  142. Pôle Bioinformatique Lyonnais, Network Protein Sequence Analysis: CLUSTALW. [http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clustalw.html]
  143. Combet C, Blanchet C, Geourjon C, Deleage G: NPS@: network protein sequence analysis. Trends Biochem Sci. 2000, 25: 147-150. 10.1016/S0968-0004(99)01540-6.PubMedGoogle Scholar
  144. Yang Z: PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci. 1997, 13: 555-556.PubMedGoogle Scholar
  145. Kent WJ: BLAT - the BLAST-like alignment tool. Genome Res. 2002, 12: 656-664. 10.1101/gr.229202. Article published online before March 2002.PubMedPubMed CentralGoogle Scholar
  146. UCSC Genome Bioinformatics. [http://genome.ucsc.edu/]
  147. EBI Sequence Analysis Tools: ClustalW. [http://www.ebi.ac.uk/Tools/clustalw/index.html]
  148. National Center for Biotechnology Information: HomoloGene. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene]
  149. Powell JR: Progress and Prospects in Evolutionary Biology: The Drosophila Model. 1997, New York: Oxford University PressGoogle Scholar
  150. FlyBase: Map Conversion Table. [http://flybase.org/static_pages/docs/cytotable3.html]
  151. Hoskins RA, Smith CD, Carlson JW, Carvalho AB, Halpern A, Kaminker JS, Kennedy C, Mungall CJ, Sullivan BA, Sutton GG, et al: Heterochromatic sequences in a Drosophila whole-genome shotgun assembly. Genome Biol. 2002, 3: RESEARCH0085-10.1186/gb-2002-3-12-research0085.PubMedPubMed CentralGoogle Scholar
  152. FlyBase: BAC In Situ Images. [http://cane.bio.indiana.edu:7062/images/lk/bac_insitu_pic/]
  153. Schalet AP: The distribution of and complementation relationships between spontaneous X-linked recessive lethal mutations recovered from crossing long-term laboratory stocks of Drosophila melanogaster. Mutat Res. 1986, 163: 115-144.Google Scholar
  154. Golic KG, Golic MM: Engineering the Drosophila genome: chromosome rearrangements by design. Genetics. 1996, 144: 1693-1711.PubMedPubMed CentralGoogle Scholar
  155. Qian S, Hongo S, Jacobs-Lorena M: Antisense ribosomal protein gene expression specifically disrupts oogenesis in Drosophila melanogaster. Proc Natl Acad Sci USA. 1988, 85: 9601-9605. 10.1073/pnas.85.24.9601.PubMedPubMed CentralGoogle Scholar
  156. Reed B: The genetic analysis of endoreduplication in Drosophila melanogaster. PhD thesis. 1992, University of CambridgeGoogle Scholar
  157. Kongsuwan K, Dellavalle RP, Merriam J: Deficiency analysis of the tip of chromosome 3R in Drosophila melanogaster. Genetics. 1986, 112: 539-550.PubMedPubMed CentralGoogle Scholar

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