comparative genomics and proteomics of drosophila, brenner's nematode, and arabidopsis:...

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Russian Journal of Genetics, Vol. 38, No. 8, 2002, pp. 908–917. Translated from Genetika, Vol. 38, No. 8, 2002, pp. 1078–1089.Original Russian Text Copyright © 2002 by Bogdanov, Dadashev, Grishaeva.

By the end of 2001, the genomes of four modeleukaryotic organisms as well as the human genomewere completely sequenced. The first eukaryotic organ-ism whose genome was completely sequenced was uni-cellular budding yeast

Saccharomyces cerevisiae

[1]. Itwas followed by the nematode

Caenorhabditis elegans

[2], the fly

Drosophila melanogaster

[3, 4], and the“botanic Drosophila,” the cruciferous plant

Arabidop-sis thaliana

[5]. These advances provided abundant evi-dence for comparative analysis of genomes and proteinsets (proteomes). Specialized fields of knowledgedeveloped that had all characteristics of an establishedscience, i.e., specific problems and methods of investi-gation. Specific branches of these sciences, compara-tive genomics and comparative proteomics, appeared[6, 7]. The latter areas of research are closely relatedbecause information on the complete DNA structure ofgenes serves as basis for the computer-aided predictionof the protein primary structure. The reverse task, geneidentification on the basis of the protein primary struc-ture, can be also solved. Special computer programssuch as BLASTP, etc. [6, 7] were developed for thesepurposes. Comparison of the genomes and proteomesin the organisms listed above is obviously important inthe context of the general theory of evolution.

SOME GENERAL PRINCIPLES OF COMPARATIVE GENOMICS

AND PROTEOMICS

At the early stage of development of comparativegenomics and comparative proteomics, researchers

have reasonably set a simple task: to identify similargenes and proteins in organisms of different evolution-ary complexity. The concepts of orthology and paral-ogy for genes and proteins have been formulated.

Orthologs are genes and proteins of different speciesthat fulfill a similar function and are closely homolo-gous in primary structure, which theoretically indicatestheir common ancestry. In comparative analysis, theorthologs are usually grouped into clusters and familiesof genes or proteins. These clusters form branchingphylogenetic trees. According to the evolution theory,orthologs are conserved genes and proteins. In chromo-somes, orthologous genes from different gene familiesexhibit synteny, i.e., linear linkage with other genesassigned to other families of the conserved gene, whichis a phenomenon of apparent physiological signifi-cance. These syntenic groups are conserved within thelarge taxons up to the taxonomic type. In other word,the genome organization into blocks is observed, whichmay be an important factor of evolution.

In contrast to orthologs, the paralogs are nucleotideand amino acid sequences differing in function, whichprobably diverged from a common ancestor throughduplications and other rearrangements.

Comparative analysis of the completely sequencedgenomes and proteomes of the first three eukaryoticorganisms—yeast, nematode, and

Drosophila

—raiseda question of how many proteins are shared by thesethree species? Analysis of the proteins predicted byusing computer software showed that about 35% of

Drosophila

genes have putative orthologs in the nema-tode genome [7]. The search was based on the homol-

Comparative Genomics and Proteomics of

Drosophila,

Brenner’s Nematode, and

Arabidopsis

:Identification of Functionally Similar Genes and Proteins

of Meiotic Chromosome Synapsis

Yu. F. Bogdanov, S. Ya. Dadashev, and T. M. Grishaeva

Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991 Russia; fax: (095)132-89-62; e-mail: [email protected]

Received February 6, 2002

Abstract

—The published principles of computer analysis of genomes and protein sets in taxonomically distanteukaryotes are expounded. The authors developed a search strategy to identify in genomes of such organismsgenes and proteins nonhomologous in primary structure but having similar functions in cells dividing by mei-osis. This strategy based on the combined principles of genomics, proteomics, and morphometric analysis ofsubcellular structures was applied to a computer search for genes encoding the proteins of synaptonemal com-plexes in genomes of

Drosophila melanogaster

, the nematode

Caenorhabditis elegans

, and the plant

Arabidop-sis thaliana.

These proteins proved to be functionally similar to their counterparts in yeast

Saccharomycescerevisiae

(protein Zip1p) and mammals (protein SCP1).

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COMPARATIVE GENOMICS AND PROTEOMICS 909

ogy of the primary structure on the condition that theprimary structure of the entire protein molecule was atleast 80% homologous with that of the entire predictedprotein of the other organism. In that case, the proteinswere considered orthologs. The estimate of 35% is min-imal because some known orthologs were omittedbecause of molecule size requirements. In particular,these orthologs are homeodomain proteins, which arelittle similar beyond the bounds of homeodomains. Thenumber of orthologous protein pairs did not apprecia-bly decrease upon toughening the demands of similar-ity. This suggests that researchers actually identifiedorthologous proteins with a common function.

About 23% of

Drosophila

proteins have the anno-tated orthologs among proteins of the two other organ-isms, yeast and nematode [7]. The functions of thecommon proteins are most likely the same in cells of alleukaryotes.

Simultaneous protein comparison in the threeorganisms provides a complex pattern, which is diffi-cult to analyze without the simpler previous experience.Therefore, in what follows, we briefly review earlierdata on comparative analysis of genomes of the twoorganisms, unicellular yeast and the simply organizedmulticellular organism, nematode

C. elegans.

The bodyof this worm consists of as little as 950 cells, of whichapproximately 700 are somatic and the remaining onesbelong to the reproductive system.

The proteins of

S. cerevisiae

and

C. elegans

weretotally compared by a joint team of researchers fromStanford and Boston Universities and from NationalBiotechnological Center in Bethesda (United States).In total, 6217 and 19 099 open reading frames (ORFs)have been studied in yeast and nematode, respectively;information is available in Saccharomyces Database athttp://genome-www.stanford.edu/Saccharomyces/help/worm/W-Y comparison.html. This study producedunexpected and interesting results. In the organismsexamined, 57% of ortholog proteins were unique pro-teins: one protein in each organism [7]. In addition, aset of highly conserved proteins in each of these phylo-genetically distant organisms was encoded by a fewORFs: 40 and 20% in yeast and nematode, respectively(Table 1). These proteins comprise the first comparisongroup. They are involved into core cellular processes

similar in these organisms: intermediate metabolism,DNA and RNA metabolism, protein folding, intracellu-lar motion of the molecules and their degradation [6].

Analysis of the orthologous protein groups providedanother result, which was more expected by the authors[6]. The regulating proteins and signaling pathwaysproved to be far more complex in the multicellular thanin unicellular organisms. A comparison of the proteinclusters in yeast and nematode showed that althoughthere were common proteins involved in regulation andsignal transduction, their number was increased in themulticellular organisms, because of a gradual change ofdomains resulting from their shuffling [7]. That obser-vation is easily explained by the fact that multicellularorganization requires cell specialization, export andimport of cellular proteins, and development of inter-cellular communications. By comparison of differentproteins in the yeast and nematode, the authors pre-dicted some trends in evolution from uni- to multicellu-lar organisms, such as (1) evolution of novel regulatoryand signaling domains, (2) evolution in the mode ofprotein assembly from the former protein domains, and(3) expansion of some families of protein domainsthrough a series of duplications.

Another type of protein comparison groups in theyeast and nematode comprised 560 protein groups(Table 1) with more than two proteins per group, i.e.,with at least one ortholog in any of the organisms exam-ined (not necessarily in the nematode, which in generalis characterized by a larger number of proteins). Thesewere such proteins as RNA-polymerase and C-subunitsof the DNA replication factor. The latter form clusterscontaining 12 proteins. However, on the whole, at thesignificance level of 1

×

10

–50

that accounts for 80%homology in the primary structure, the number of pro-teins involved in DNA and RNA metabolism is only10% higher in the nematode than in the yeast, which, inview of the universality of major molecular biologicalphenomena (genetic code, replication, etc.), was anexpected result. In

S. cerevisiae

and

C. elegans,

theorthologs participating in DNA and RNA metabolismcomprise 18% of all proteins, which are at least 80%homologous (Table 2).

In the yeast and nematode, the number of proteinswith similar function depends much on the type of this

Table 1.

Conserved amino acid sequences in proteins of yeast and nematode

Probabilityof random homology,

P

Groups of sequences Open reading frames (ORFs)

total one memberper organism

more than one mem-ber per organism

yeast(

n

= 6217) nematode

(

n

= 19099)

1

×

10

–100

236 157 79 330 (5.3%) 370 (1.9%)

1

×

10

–50

552 322 230 888 (14.3%) 1094 (5.7%)

1

×

10

–10

1171 611 560 2497 (40%) 3653 (19%)

Note: The results were obtained by reciprocal comparison of each yeast ORF with all ORFs of nematodes and vise versa using the BLASTPprocedure. The numbers of coincidences were combined at the given probability

P

according to [6].

910


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et al

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function. As for structural cell proteins (e.g., those ofcytoskeleton) 2.1 orthologous proteins in the nematodecells on average fall to each protein in a yeast cell. Notethat we mean the number of protein denominationsrather than their bulk. In total, the orthologous cytosk-eleton proteins comprise only 5% of all proteins whichare 80% homologous in the two organisms, whereas theproteins with unknown function comprise 8% of allproteins at the same level of homology (Table 2). Theoverall physical mass of cytoskeleton proteins is greaterthan that of the proteins regulating DNA and RNAmetabolism. However, this topic is beyond the scope ofproteomics but rather a subject of cell biology.

The above quantitative and qualitative analysis atthe level of the entire genome comparison confirmedthe evidence obtained previously by other methods, i.e.,the fact that approximately the same number of orthol-ogous proteins is involved in the core molecular geneticprocesses in phylogenetically distant organisms. Inaddition, it was found that the threefold difference inthe total amount of proteins with similar functionsrevealed in yeast and nematode cannot be attributed tothe variability of the protein clusters, although substan-tial variability has actually been detected. The point isthat both yeast and nematode have species-specific pro-teins. In particular, sets of such proteins associated withintracellular signal transduction (humoral, ionic) werefound [7]. Such conclusions are not new: they weredrawn with respect to individual cell functions longbefore the advent of the age of genomics.

Thus, analysis of the genomes showed that the com-parable number of proteins similar in structure mediatemajor biochemical processes in the unicellular

S. cere-visiae

and multicellular

C. elegans.

Hence, the conclu-sions inferred from protein analysis in some organism(the first organism subjected to such an analysis was

yeast) can be applied to another organism, in which theorthologs were detected from the primary structure.Comparative analysis of the proteins of core metabo-lism in yeast and nematode provided results that agreewith these views.

Along with identification of orthologs from homol-ogy of the primary structure, structural orthologs can bealso identified as the proteins with common domainsand similar domain organization. In contrast to the firstapproach based on evolutionary considerations, the lat-ter is a structural–functional approach based on the rea-soning that the functions of the structurally similar pro-teins are the same in different organisms. The proteinsassembled from several domains often contain at leasttwo readily identifiable domains, which are similar orhomologous in different organisms. Thus, a domain ofthe eukaryotic protein kinase (access no. IPR 000719)is shared by 199 and 388 proteins of

S. cerevisiae

and

C. elegans

, respectively; the zinc fingers domain typeC2H2 (access no. IPR 001304) is encountered 47 and138 times in different proteins of these organisms,respectively. Comparative computer analysis of theseproteins successfully employed such databases as Inter-Pro (http://www.ebi.ac.uk/interpro) and NCBI CDART(http://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi? cmd=rps). Analysis by InterPro supple-mented by “manual” analysis revealed 1400 proteinfamilies and individual domains, of which 984, 1133,and 1177 ones were identified in yeast, nematode, and

Drosophila,

respectively. Note that 744 families ordomains were shared by these three organisms [7].Hundreds of proteins in yeast, nematode, and

Droso-phila

have similar domain organization and participatein cytoskeleton formation, regulation of cell cycle, celladhesion, and other intra- and intercellular structuresand processes.

What is the practical value of this study? The mostimpressive especially for the government and science-financing organizations, was the fact that among289 genes for human diseases, which had been alreadystudied and available for rapid analysis, 177 and150 genes had orthologs in

D. melanogaster

and

C. ele-gans

, respectively [7]. Thus, many problems, such asmolecular biology of these genes, their phenotypicexpression, and interaction with other genes can bestudied with the more accessible and inexpensive mod-els—

D. melanogaster

, nematode, and even yeast [8].The first example of this kind was the experiments con-ducted as early as in 1985, when the mammalian signal-ing protein RAS was substituted by an orthologous pro-tein in RAS-deficient yeast strains [7].

As far back as in 1926, Timofeeff-Ressovsky andVogt substantiated the applicability of genetic and espe-cially phenogenetic results obtained on

Drosophila

forrecognizing genetic mechanisms of human diseases[9]. This idea was repeatedly discussed by Muller, aclassic of genetic studies on

Drosophila

[10]. Theresults of comparative analyses of genomes and pro-

Table 2.

Percentage of families of the conserved proteins(orthologs) with at least 80% homology of the primary struc-ture in yeast

S. cerevisiae

and nematode

C. elegans

at thethreshold of similarity level,

P

= 1

×

10

–50

(based on data byChervitz

et al

. [6])

FunctionPercent

of all con-served proteins

Ratio of the protein number

in nematodeto that in yeast

Intermediate metabolism 28 1.0

DNA and RNA metabolism 18 1.1

Protein foldingand degradation

13 1.2

Transport and secretion 11 1.5

Signal transduction 11 1.2

Unidentified proteins 8 1.1

Ribosome proteins 6 0.6

Cytoskeleton 5 2.1


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teomes in the model organisms, which were publishedin 2000, initiated the overall cataloging of evidenceallowing comparative genomics to be used in solvingthe medical and genetic problems [6, 7].

FUNCTIONALLY SIMILAR BUT NONHOMOLOGOUS GENES

AND PROTEINS IN PHYLOGENETICALLY DISTANT EUKARYOTES

At the present stage of the development of biologi-cal sciences connected with the genome and the cell, anew problem can be formulated, which is at the bound-ary of genomics and proteomics, on the one hand, andcell biology, on the other. The point is that in dividingcells of phylogenetically distant organisms, there areintracellular structures with similar functions thoughwith partially or completely different ultrastructure.These are kinetochores of chromosomes, cell centers(animal centrioles, spindle polar bodies in fungi, andamorphous cell centers of plants), and the synaptone-mal complexes playing the key role in synapsis andgenetic recombination of homologous chromosomesduring meiosis. All these structures account for thechromosome behavior during meiosis, their segregationto the poles of a dividing cell (kinetochores and cellcenters) or formation and fixation of pairs of homolo-gous chromosomes (synaptonemal complexes).

In different kingdoms of eukaryotes, theseorganelles differ in ultrastructure suggesting that inphylogenetically distant taxons they might be assem-bled from different structural proteins. Among the lat-ter, the orthologous proteins may be either absent,which would be a clearly obvious phenomenon, orpresent, which would be an additional evidence of theorganic world unity.

We had faced this problem in 2000, when analyzingand comparing specific genes of meiosis in

D. melano-gaster

,

S. cerevisiae

, and other organisms [11, 12]. Wewere interested in genes encoding the proteins of thesynaptonemal complex (SC).

The formation of the SC superstructure is observedonly during meiotic prophase I in most eukaryotes toplay an important role in meiosis [13]. Mutations ofgenes responsible for the SC formation lead to a partialor complete inability of chromosomes to form pairs ofhomologs prior to or during crossing over as well as tosuch phenomena as reduced frequency or completeelimination of crossing over, achiasmaty, and disturbedsegregation of homologous chromosomes during meio-sis I. As a consequence, either aneuploidy or sterility ofgerminal cells is observed.

The scheme of the SC formation is the same in alleukaryotes [13]. The meiosis-specific SC consists ofthe two protein axes along each homologous chromo-some (lateral SC elements), which are positioned inparallel and connected by numerous transverse proteinfilaments (Fig. 1). The space between the lateral ele-

ments is referred to as the central space. The chromatinfibrils are bound to the lateral elements. Thus, the SCserves as a frame temporarily holding homologouschromosomes in a strict order so that the homologousloci occurred one opposite of the other. The centralspace of the SC is occupied by recombination nodules,which are conglomerates of enzymes necessary forDNA recombination [13]. Ascomycetes, nematodes,insects, higher plants, mammals, and organisms ofother taxa share the general structure of the SC but itsdetails vary among these taxa. The structural SC pro-teins (those of the lateral elements and transverse fila-ments) are studied only in the four mammalian species[14–17] and in yeast

S. cerevisiae

[18, 19] (see [20] and[21] for review). In yeast and mammals, these function-ally similar proteins showed no homology in their pri-mary structure. This raises a question of how the func-tionally similar cells, namely the cells enteringprophase of meiosis, create functionally similar subcel-lular structures from different proteins in phylogeneti-cally remote organisms? What are the traits of the SC-forming proteins which are decisive for the SC assem-bly according to the same scheme in different types andeven kingdoms of eukaryotes?

We proposed that the proteins of the transverse fila-ments in the central SC space are the simplest for suchcomparative analysis. In yeast and mammals the singleprotein containing about 900 amino-acid residues con-stitutes transverse filaments. This protein consists ofthe three domains: the central one with an extendedcoiled coil and two terminal globular domains incapa-ble of forming the coiled coil [18].

Combined immunocytochemical analysis and elec-tron microscopy showed that two pairs of molecules ofthese proteins form the transverse filaments of the SC[22] (Fig. 2). In each pair, the molecules lie in paralleland are similarly oriented in the central space of the SC;

TF

CS

CE LE Chr

RN

Fig. 1.

Scheme of the synaptonemal complex. LE, lateralelements; CS, central space; CE, central element; TF, trans-verse filaments involved into CS formation; Chr, chromatinloops; and RN, recombination nodules.

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they are perpendicular to the lateral elements andform a tandem. The protein molecules are head-to-head(N-terminus-to-N-terminus) oriented. Their C-terminican bind DNA and are embedded into the lateral ele-ments of the SC. The width of the central SC spacedepends on the size of the central rod-shaped domain.Deletions in the central domain revealed in the allelicmutants for the

ZIP1

yeast gene were found to diminishthe width of the SC central space to bring the lateral ele-ments together [18, 19].

Based on the literature data we performed correla-tion analysis for two traits: the width of the SC centralspace and the size of the protein molecules forming thetransverse SC filaments. A high correlation (

r

= 0.85;

P

< 0.001) was found between the width of the centralSC space and length of the entire SCP1 and Zip1p pro-tein molecules in mammals and yeast, respectively(Fig. 3). The correlation between the length of the cen-tral coiled-coil protein domains and the width of the SCcentral space was even higher (

r

= 0.90;

P

< 0.001).Such a high and significant coefficient of correlationsuggests a functionally important dependence, i.e., thewidth of SC central space directly depends on thelength of the central (linear in shape) domain of the pro-teins SCP1 and Zip1p constituting the transverse fila-ments [23].

These findings allowed us to undertake a computersearch for

D. melanogaster

protein similar to the yeastZip1p and mammalian SCP1 proteins. We proposedthat the putative protein of

D. melanogaster

could alsobe assembled from the three domains and its centraldomain contained a coiled coil of the length equal to ahalf of the SC central space in

D. melanogaster.

Natu-rally, we primarily analyzed those proteins which arespecifically expressed in meiosis.

Computer Identification of the SC Gene and Protein in D. melanogaster

The computer-aided search for orthologs of theknown proteins is based on a comparison of their pri-mary structure even if the data on the secondary struc-ture (domain organization) are used. Thus, the com-puter procedure SGD Worm–Yeast Protein Compari-son (http://genome-www.stanford.edu/Saccharomyces/worm/) is based on a comparison of amino acidsequences with known consensus sequences of the pro-tein families and protein domains. However, in thisway, both structural and functional analogs are uniden-tifiable in the phylogenetically distant organisms,because the role of these proteins in the formation ofsubcellular structures in the meiotic cells is not takeninto account. Therefore, we employed anotherapproach, when the results obtained

in silico

(fromcomputer databases) are used in combination withthose of the morphological and genetic experiments.Such analysis is still poorly automated and based on the“manual” comparison of the secondary structures (forexample, the length of the coiled-coil region) in the pro-teins of interest.

Among more than 80 genes controlling meiosis in

D. melanogaster

[12], only one,

c(3)G

(

crossover sup-pressor on 3 of Gowen

) located in the 89A2–5 region ofthe cytological chromosome map [24] is undoubtedlyinvolved specifically in the SC formation [25]. How-ever, neither the mechanism of its effect nor the relevantgene product were identified until 2001 [26]. One of theold hypothesis suggested that

c(3)G

may encode one ofthe SC components [25]. We aimed to analyze in

D. melanogaster

the virtual products of genes from the3R chromosome region, which excessively overlaps the

c(3)G

locus. For this purpose, we studied all geneslocalized within the 88E–89B region of the Bridges

20

nmLE CE LE

Fig. 2.

Scheme of the central space of the synaptonemalcomplex (SC). Designations: dotted line, borders of chro-matid axes within SC lateral elements; open rectangles, theterminal (globular) domains of protein constituting thetransverse filaments; gray rectangles, central (coiled-coil)domains of the transverse-filament protein; small blackrectangles, hypothesized protein–protein contacts betweenthe terminal domain. Other symbols as in Fig. 1.

20018016014012010080604020400 600 800 1000 1200 1400

The protein molecule size, a.a.

CS width, nm

Fig. 3.

Dependence of the width of the SC central space onthe size of the protein molecule involved in the formation ofthis space. CS, central space of SC; a.a., amount of aminoacid residues in the protein. Black circles indicate rat,mouse, and human SCP1 proteins and various Zip1p pro-teins from yeast

S. cerevisiae

, (both wild-type protein andmutants with internal deletions and duplications of differentlength). Slanting straight line is the regression line for theabove traits. Dotted line indicates 95% confidence intervalof the regression line.


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cytological map. In particular, we decided to comparethe domain organization and secondary structure of thepredicted

Drosophila

proteins and the known SC pro-teins in yeast and mammals. Seventy-eight annotatedgenes from region 88E6–89B2 containing about 250 kb(according to the NCBI database) were analyzed

in sil-ico.

The protein products of these genes had not beenpreviously identified. The virtual protein product ofonly one of these genes proved to be similar to theSCP1 and Zip1p proteins in size and domain organiza-tion. That was the product of the

CG17604

Drosophila

gene according to the NCBI nomenclature. The geneproduct consists of the three domains: the two terminaland one central domain with rod-shaped and coiled-coilrod-shaped structure. The

CG17604

gene is located onchromosome 3R at position 36250–36253 kb accordingto the NCBI molecular map. The virtual product of thisgene contains 744 amino acids, and its central domain(495 amino acids according to the verified data) canform the coiled-coil structure similar to that of the cen-tral domains in Zip1p and SCP1 proteins. The width ofthe SC central space in

D. melanogaster is 109 nm [27].When these values were plotted (Fig. 4), the pointobtained occurred within the 95% confidence intervalof the regression line for the following two traits: thesize of the coiled-coil region of the protein moleculesand the width of the SC central space in mammals andyeast. In D. melanogaster, the coiled-coil region of theprotein encoded by the CG17604 gene proved to beequal to a half-width of the SC central space. The samewas revealed upon comparison of the SC structure inyeast and mammals.

As judged from the primary structure [26], the pre-dicted CG17604-gene product is homologous to vari-ous proteins, many of which can form coiled coils. Inparticular, it is homologous to the NUF1 protein of thespindle polar body (S. cerevisiae), to the myosin heavychain (D. melanogaster, C. elegans, A. thaliana, Homosapiens, Dugesia japonica), and to the laminar proteins(Mus musculus). The known SC proteins (yeast Zip1p[19], rat and mouse SCP1 [14, 16]) were also homolo-gous to the above proteins. Interestingly, the amino acidsequence of the CG17604 gene product was homolo-gous to neither SCP1 nor Zip1p, as well as no homol-ogy was found between the Zip1p and the proteins ofSC filaments in mammals [19]. Thus, although thenucleotide sequence of the deduced gene CG17604 ofD. melanogaster was not homologous to that of theZIP1 and SCP1 genes, the three genes were similar inthat they were partially homologous to the genesencoding the above large family of the structural pro-teins.

In addition, we have analyzed the protein productsof D. melanogaster genes from nine regions carryingother meiotic mutations: mei-9, mei-41, mei-217, mei-218, mei-P14, mei-P22, mei-P26, mei-w68, mei-S282.These protein products were annotated by the CeleraGenomics company. None out of 200 annotated geneproducts of the above regions could form a coiled coil

with length necessary to ensure formation of transverseSC filaments in Drosophila.

The evidence obtained were additionally verified byestimating the isoelectric points (pI) of the known SCproteins from yeast and mammals, and of the CG17604gene product. Using the ProtParam computer program,both entire molecules and individual domains wereanalyzed (Table 3). Except for pI of the N-terminaldomain, all parameters of the CG17604 protein weresimilar to those of the proteins Zip1p and SCP1/SYCP1.The C-terminal domains of the above proteins having atotal positive charge (basic, i.e., alkaline character) areknown to be embedded into the lateral SC elementsand, therefore, they are in contact with DNA; whereasthe N-terminal domains with a total negative charge(acidic character) are directed into the SC central space,where they overlap and form the SC central element[16, 19]. In Drosophila CG17604 protein, the positivecharge of N-terminal domain probably accounts for thespecific morphology of SC central element with itsclearly defined striated ultrastructure and a relativelylarge width (32 nm) [27]. Thus, we have detected aproduct of the CG17604 gene, which is similar in mostparameters to the known proteins of SC transverse fila-ments of other organisms.

According to the FlyBase data, the CG17604 gene islocated in the 89A7-8 region. At the same time, thec(3)G gene, which is proposed to be identical to theCG17604 gene, was localized to the 89A2 region (Fig. 5).This discrepancy is probably caused by objective diffi-

200

180

160

140

120

100

80

60

40

20100 200 300 400 500 600 700 800 900

CS width, nm

Coiled-coil size, a.a.

Fig. 4. Dependence of the width of SC central space on thesize of the coiled-coil part of the protein molecule involvedin the formation of SC transverse filaments. Designations asin Fig. 3. Coordinates of the open circle, open oval, andrectangle correspond to the widths of SC central space andlengths of the coiled coil of the CG17604 protein of D. me-lanogaster, 781586 protein of C. elegans, and AAD10695protein of A. thaliana, respectively; coordinates of thehatched oval corresponds to the width of SC central spaceand half-length of the coiled coil of the Q11102 protein ofC. elegans.

914

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BOGDANOV et al.

culties in mapping that region of the third D. melano-gaster chromosome. This conclusion is further sup-ported by a relative shift of positions of genes in thisregion on the molecular and cytogenetic NCBI maps(Fig. 6). We suggest, therefore, that it is the CG17604gene that encodes the protein of transverse filaments ofthe central space of D. melanogaster synaptonemalcomplex and that this gene is most likely the knownc(3)G gene. That suggestion can be experimentallytested. As such a test, Hawley and coworkers have ana-lyzed the molecular organization of both the c(3)Ggene and its product independently of and simulta-neously with us [28, 29]. In Proceedings of the 17th

European Drosophila Research Conference (Septem-ber 1–5, 2001), these authors reported that the c(3)Ggene encoded a protein similar in structure to the Zip1pand SCP1 proteins [28]. In this study, the protein prod-uct of the c(3)G gene and monoclonal antibodiesagainst that protein were obtained. As shown by immu-nofluorescent microscopy, in D. melanogaster oocytesthe central part of bivalents in prophase was coloredwith these antibodies [29]. Thus, location of antibodiesagainst the protein product of the c(3)G gene coincidedwith the proposed location of the CG17604 protein,which supports our suggestion that the c(3)G andCG17604 genes are identical.

Table 3. Parameters of the molecules of proteins identified experimentally [14–19] and predicted (this study), which are in-volved in formation of SC transverse filaments in organisms with completely sequenced genome

Biological species, their normal and mutant proteins

Size of protein molecules and domains pI of proteins and domains

entirelength of the

molecule

lengthof the coiled-

coil region

widthof SC CS

N-terminal domain

centraldomain

C-terminal domain

entiremolecule

M. musculus SCP1 993 713 100 5.9 5.3 9.7 5.8

H. sapiens SCP1 973 677 100 5.0 5.4 9.7 5.7

R. norvegicus SCP1 946 717 100 4.2 5.3 9.8 5.6

S. cerevisiae Zip1 875 632 115 4.8 6.1 10.1 6.4

Zip1-m2* 583 285 63

Zip1-mc1* 484 170 49

Zip1-mc2* 776 634 101

Zip1-n1* 732 578 118

Zip1-nm1* 767 512 118

Zip1-2XH2** 1012 799 153

Zip1-3XH2** 1280 1067 189

D. melanogaster CG17604 744 495 109 10.0 4.9 9.7 5.9

A. thaliana AAD10695 921 476 100–120 5.3 5.4 9.0 5.6

C. elegans Q11102 1132 938 70–85 11.9 5.1 11.0 5.5

C. elegans T26844 1083 536 70–85 8.1 6.9 5.3 6.6

C. elegans T27907 772 460 70–85 5.2 5.9 5.7 5.1

C. elegans Z81586 484 460 70–85 4.9 9.5 10.0 9.4

C. elegans WP:CE17456 213 50 70–85

Note: CS, central space of SC; pI, isoelectric point of a protein (domain); *, yeast Zip1p proteins with deletions; **, the same proteins withduplications.

89 A 89 B

spno Aldox-1 tbi CG5614 spn-E glob1

Poost

CG5404 CG18505 ND23 CG4224CG4560 CG14877

CG14875 CG4225CG17604

recc(3)G

Fig. 5. Genetic surrounding of the CG17604 gene according to the FlyBase data. Positions of the annotated genes revealed by com-puter-aided methods (including the CG17604 gene) and positions of some genes determined by genetic methods (including thec(3)G gene) are indicated on a cytological map of Drosophila chromosome 3 in region 89A–89B.



A Computer Search for Genes and Proteins of Synapsis in A. thaliana

A search for specific sequence of the c(3)G gene inDrosophila was limited by a relatively small regionand, therefore, a small number of the putative geneshave been analyzed (78 genes). In other organisms, thegenomes of which were completely sequenced, detec-tion of genes with function similar to that of genesSCP1 and ZIP1 was complicated by a lack of mutationsleading to disturbed formation of the synaptonemalcomplex. In these organisms, the region of search wasexpanded to the entire genome. To reduce the numberof proteins examined, one more stage of analysis wasnecessary, namely, a search for the proteins withdomain structure typical of the SCP1 and Zip1p pro-teins. We used the CDART (NCBI) software program(http://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi?cmd=rps) to assess the secondary struc-ture of analyzed proteins and to reveal those havingdomain organization similar to that of either Zip1p orSCP1 and the size adequate for the formation of SCtransverse filaments in A. thaliana.

To our knowledge, there are no published data onthe width of SC central space in A. thaliana. Neverthe-less, analysis of literature revealed an important featureof the general SC organization of most phylogeneticallydistant organisms, including higher plants: the width ofthe central space is at most 90–120 nm. Based on theseconsiderations we detected a virtual protein AAD10695in A. thaliana proteome, which was similar to the Zip1pand SCP1 proteins in size and domain organization(Table 3, Fig. 4). As mentioned above, the importantfeature of the known proteins of SC transverse fila-ments is the basic (pI > 8) character of their C-terminaldomain. This was characteristic of the AAD10695 pro-tein of A. thaliana.

Thus, like in D. melanogaster, only one putativefunctional analog of the mammalian SCP1 and yeastZip1p was revealed in A. thaliana, which is in agree-ment with the major evidence of comparative proteom-ics concerning highly specialized proteins.

Specific Features of SC Genes and Proteinsin Nematode C. elegans

In C. elegans, several putative proteins annotated asorthologs for the Zip1p protein were identified in silicoprior to our study. For instance, according to the Worm-Base data (http://www.wormbase.org/), the WP:CE17456protein, a product of the syp2 gene, is a componentof the SC central space in C. elegans [30]. Using theProtParam software program (http://www.expasy.ch/tools/protparam.html) the amino-acid sequence ofthis protein was analyzed. The results obtained sug-gested that the WP:CE17456 protein is unable to forma coiled-coil structure with a length adequate (Table 3)for the formation of SC transverse filaments. Webelieve, therefore, that this protein does not constitute

the SC transverse filaments. According to the data ofProteome Inc. (http:/www.proteome.com/index.html),the protein Z81586 of C. elegans is also an analog ofthe SCP1. Indeed, the Z81586 may be a functional ana-log of the latter as judged from the molecule size,domain organization, the size of the coiled-coil centraldomain, and physicochemical properties. However, thepoint is that in C. elegans the SC morphology differsfrom that in yeast, mammals, and Drosophila. The dis-tinctive feature of the SC in C. elegans was a lack of aclearly defined central element. In other organisms, thecentral element had an appearance of an electron-denseband in the central space lying in parallel to the lateralSC elements [31]. In some photomicrographs of the SCfrom C. elegans made by electron microscopy ofultrathin sections, the transverse filaments did not crossany central element on their way from one to the otherlateral element of SC [32], which suggests a differenttype of molecular organization of the central space inC. elegans. In other organisms, two molecules directedtowards each other are involved in the formation of thetransverse filaments; they interact by their N-termini toform a central element (model 1). In contrast, in C. ele-

~ ~

~~ ~

1 2 3

88F

89A

89B

Surf4

Act88F

ENL/AF9spnol(3)rN346c(3)G

l(3)89Acl(3)89AaScp2T-A7.1Mat89Bb

l(3)S079311l(3)neo46dkn

88F3-88F4

88F7-88F7

88E-88F89A1-89A189A1-89A289A2-89A2

89A2-89A5

89A2-89A589A-89A89B1-89B1

89B-89B

89B-89B89B1-89B689B-89B

Genes Positionon the cytologic

map

Fig. 6. Cytogenetic and molecular maps of the 88F–89Bregion in D. melanogaster chromosome 3, where the c(3)Ggene is localized according to NCBI data. The known genesand their positions are indicated on the cytological map:1, cytological map; 2, gene positions on the molecular map;3, gene positions on the cytogenetic map. Thin lines connectthe sites of gene location on the two maps. Discrepancybetween the molecular and cytogenetic maps for the studiedregion appears as a shift of the relative gene positions on thetwo maps.

916


BOGDANOV et al.

gans, the single long molecule probably extends fromone to the other lateral SC element (model 2). If this isthe case, both terminal domains of the protein should bebasic. Schmekel and Daneholt proposed this mode oforganization of the SC central space in the beetle Blapscribrosa [33].

In view of the above, we have undertaken our ownsearch for the analogs of the SCP1 and Zip1p amongthe proteins of C. elegans. Note that we selected pro-teins corresponding to both models: model 1 of two rel-atively short proteins positioned head-to-head andmodel 2 of only one long protein (Table 3). In thegenome of C. elegans, we found three genes encodingsuch proteins. All these proteins (Q11102, T26844, andT27907) contained coiled coils of the adequate size.With regard to the Q11102 protein, which fits the model2, a half-length of the coiled coil was taken, whereas thefull-length coiled coils were taken for the two remain-ing proteins. Only the Q11102 protein met the require-ment of a basic isoelectric point (pI) of the terminaldomains according to model 2 (long proteins whoseboth terminal domains are basic). On the other hand,the only protein that fits model 1 (Fig. 4) by all param-eters was the Z81586 protein. To decide between thetwo proteins, we used the Worm Base software to ana-lyze the genetic surrounding of the two candidate genesencoding proteins Z81586 and Q11102, namely,the genetic map regions –4 ± 1 of chromosome 1 and–10 ± 3.5 of the X chromosome, respectively. None ofthe known genes responsible for normal meiosis wasdetected in these regions. Thus, we are still unable todecide between the two proteins. However, the uncom-mon morphology of the SC central space in C. eleganssuggests that the Q11102 protein is the functional ana-log of the SCP1 and Zip1p proteins and that the trans-verse SC filaments in this nematode are formed by thelong molecules of this protein, which spans the entirecentral space in contrast to yeast and mammals (in thelatter, the SC central space is formed by two proteinmolecules directed towards each other).

CONCLUDING REMARKS

Total genome sequencing in the three model objects,yeast, nematode, and Arabidopsis, as well as the devel-opment of special computer programs allowedresearchers not only to reveal the open reading framesof previously unknown genes, but also to predict thestructural and functional parameters of the appropriateprotein products. The two basic methods of an auto-mated search for orthologs have limitations and can beused only for preliminary selection (except in someobvious cases). We have proposed an additional methodof accurate identification of protein analogs in phyloge-netically distant organisms. It consists in selection fromcomputer databases of the proteins whose secondarystructure corresponds to the spatial ultrastructuralparameters of cell organelles. This approach proved tobe productive, when applied to the key protein of the

synaptonemal complex in the three taxonomically dis-tant organisms. The adequacy of this method was sup-ported by the experiments on detection of this proteinin Drosophila. It remains to be seen whether thisapproach is productive when applied to other proteinsconstituting the intracellular structures of other organ-isms.

Of interest is the task to establish the mode of for-mation of the synaptonemal complexes and other cellstructures assembled from proteins that have no homol-ogy in the primary structure but are structurally analo-gous in different kingdoms of eukaryotes [34].

ACKNOWLEDGMENTS

This work was supported by the Russian Founda-tion for Basic Research (project nos. 99-04-48182 and02-04-48761).

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