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MAIK “Nauka

/Interperiodica”0090

Russian Journal of Genetics, Vol. 38, No. 1, 2002, pp. 90–94. Translated from Genetika, Vol. 38, No. 1, 2002, pp. 108–112.Original Russian Text Copyright © 2002 by Bogdanov, Grishaeva, Dadashev.

Cell divisions (meiosis and mitosis) are controlledby general and specific genes. In

Drosophila melano-gaster

, more than 80 specific meiotic genes have beenfound [1]. One of key specific meiotic genes, which isthe earliest with regard to the time of action, was named

crossover suppressor of Gowen

c(3)G

.

c(3)G

mutantslack crossing over and the synaptonemal complex (SC)and exhibit chromosome nondisjunction [2]. The geneproduct and mechanism of action of this gene are notknown [3]; according to one hypothesis,

c(3)G

codesfor an SC component [2].

The SC is formed in meiosis in most of highereukaryotes; its formation is associated with synapsisand crossing over of homologous chromosomes [4].Mutations leading to SC anomalies considerably affectsynapsis and recombination. Interference of crossingover directly depends on the SC presence [5]. In thepast decade, the molecular organisation of SC hasbecome a focus of meiosis studies. Specific SC proteinsand their genes has been established in two models:yeast

Saccharomyces cerevisiae

and mouse

Mus mus-culus

[6–9]. Due to the histological properties ofgonads and cytological features of meiosis in

Droso-phila,

the SC cannot be isolated in preparative quanti-ties. The corresponding proteins also cannot be isolatedbecause in

Drosophila

females, the SC is formed onlyin two cells of the 16-cell cluster and males of thisorganism lack the SC [10]. Consequently, we have cho-sen another method of search for putative protein C(3)G,namely, computer screening of the

Drosophila

genomefor a structural gene whose virtual product was similar

to the known structural SC proteins in yeast and mam-mals. For the screening, we used the complete data baseof

D. melanogaster

genome—FlyBase [3].

We have chosen the following pathway of C(3)Gidentification: from the ultrastructural characteristics ofthe SC in

Drosophila

and other organisms to the struc-tural characteristics of the known proteins in yeast andmammals and further to virtual proteins whose genesare located in the localization region of the

c(3)G

gene.

Our attention was focused on the ultrastructure ofthe SC central space because mutations of genes codingfor the single meiotic structural protein of the SC cen-tral space in yeast, Zip1, causes asynapsis, i.e., theinability to form the SC from the precursor axial coresof the prophase meiotic chromosomes. Protein Zip1 of

S. cerevisiae

and the functionally analogous SCP1(SYCP1) protein of mammals form transverse fila-ments in the SC central space and link synaptic homol-ogous chromosomes. The width of the central space,i.e., the distance between synaptic homologs, is ratherconservative in a wide variety of organisms [11] consti-tuting 90–120 nm in fungi, 100–120 nm in insects, about100 nm in mammals [11, 12]. The central space is filledwith transverse protein filaments. The secondary struc-ture and conformation of proteins forming the trans-verse filaments was established in yeast (Zip1) [7, 8],rat (SCP1) [9], mouse [6], and hamster (SYN1 [13]. Inhumans, a homolog of rat SCP1 was found [14]. Allthese proteins are characterized by similar features dis-tinguishing them from other proteins. Their moleculesconsist of three domains. The central domain forms a

Gene

CG17604

of

Drosophila melanogaster

May Be a Functional Homolog of Yeast Gene

ZIP1

and Mammal Gene

SCP1

(

SYCP1

) Encoding Proteinsof the Synaptonemal Complex

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

Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991 Russia; fax: (095) 135-12-89; e-mail: dadashev@vigg.ru

Received August 8, 2001

Abstract

—From data on the molecular organization of transverse filament proteins of the synaptonemal com-plex (SC)—Zip1 in yeast and SCP1 in mammals—and on the width of the SC central space in these organismsand in

Drosophila

, the putative molecular structure and size of a transverse filament protein of the SC in

Droso-phila

has been inferred. Using genetic and molecular databases and software from the Internet, we carried out

in silico

screening for a candidate gene for the

Drosophila

transverse filament protein. As a most likely candidate,gene

c(3)G

was chosen. The search in the 250-kb region overlapping the locus of this gene (sections 88E-89B)and containing 78 predicted genes has revealed only one gene,

CG17604

, whose protein meets all requirementsfor the transverse filament protein of the SC. It was suggested that gene

CG17604

is gene

c(3)G.

In this case,gene

c(3)G

must be localized in section 89A7-8 of the cytological map of

Drosophila melanogaster.

SHORTCOMMUNICATIONS

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rod-shaped coiled coil whereas N-terminal (acidic) andC-terminal (basic) domains have the globular structure.Each of the transverse filaments of the SC consists oftwo overlapping protein dimers (Zip1 in yeast or SCP1in mammals) in the “head-to-head” (N-end to N-end)orientation [6–8]. The C-terminal domains, which haveDNA-binding activity, attach to the DNA-containinglateral SC elements [7]. Comparisons of the primarystructure of these proteins and corresponding genes inmammals and yeast did not reveal homology but 74–93% homology was found among SCP1/SYN1 proteinsof various mammal species [8]. SPC1/SYN1 and Zip1exhibit homology with the proteins of the heavy myo-sin chain, nuclear lamina, nuclear matrix and some oth-ers [6–9,13, 14]. In

Drosophila

, no SC proteins havebeen found so far [3].

To search for the putative protein product of

c(3)G

,we used computer databases and information resourcesfrom the Internet (Table 1).

Gene

c(3)G

has been localized in the 89A2-5 regionof the cytological map of

D. melanogaster

[3, 15]. Thisregion is rich in mutations affecting viability and fertil-ity. All of these mutations are expressed exclusively inthe female germline cells [15]. In addition to

c(3)G,

they include meiotic mutation

rec.

We have examined78 annotated genes from the NCBI database found inthe localization region of the

c(3)G

gene and in adja-cent sections (88E6 through 89B2) whose gene prod-ucts had not been identified.

We have preliminarily examined the relationshipbetween the width of the SC central space in yeast andmammals and the length of the protein molecules form-ing the transverse SC filaments. This analysis was car-ried out on the basis of literature data [6–9, 13, 14] andincluded data on yeast mutants carrying particular dele-tions and duplications of the Zip1 protein [7]. Theobtained coefficients of correlation between the widthof the SC central space and the length of the proteinmolecules (

r

= 0.85,

P

≤

0.001; Fig. 1) and especiallythe length of the central (rod-shaped) domain of theprotein molecule (

r

= 0.9,

P

≤

0.000) were high.Since the width of the SC central space in

Droso-phila

is about 110 nm [10], the length of the sought

gene product C(3)G must be within the limits of 700 to1000 amino acids.

The size of the examined region was about 250 kb.Gene products of the sizes smaller than 500 aminoacids were not taken into analysis because a prelimi-nary correlation analysis (Fig. 1) allowed us to limit thearea of search to 730–970 amino acids.

Using the ISREC software procedure, we haveanalyzed the secondary structure of the virtual geneproducts of

Drosophila.

It was shown that the virtualproduct of only one gene can form a coiled coil of therequired length. Only the central part of this virtualprotein has the coiled coil structure whereas its N- andC-end domains are globular. The corresponding

CG17604

gene is located on chromosome 3 at posi-tion 36250–36253 kb according to the NCBI molecu-lar map.

200

400

SC central space width

Number of amino acids in protein molecule

160140

100

180

120

80604020

600 800 1000 1200 1400

Fig. 1.

The relationship between the SC central space widthand size of the protein molecule forming transverse filamentsin SC central space. Solid circles correspond to proteins SCP1(human, rat, mouse) and Zip1 with internal deletions andduplications of various length in yeast

S. cerevisiae.

The slop-ing solid line shows the regression for the given characters.The dashed lines show the confidence limits of regression atthe 95% confidence interval. The horizontal line indicates theSC central space width in

Drosophila;

the expected number ofamino acids in the protein molecule is limited by vertical lines.

Table 1.

Names, characteristics, and addresses of the Internet informational resources used

Name Characteristics Address

FlyBase: A Database of the Drosophila Genome (GadFly: Genome Annotation Database of Drosophila)

Drosophila

database (mutations, nucle-otide sequences, etc.)

http:///flybase.bio.indiana.edu/

NCBI (National Center for Biotechnology Information)

Molecular biology, biochemistry,and genetics

http://www.ncbi.nlm.nih.gov/

ExPASy Molecular Biology Server(Expert Protein Analysis System)

Program for predicting physicaland chemical protein properties(ProtParam)

http://www.expasy.ch/tools/protparam.html

BCM Search Launcher: ProteinSecondary Structure Prediction

Program for predicting the secondary protein structure (ISREC)

http://dot.imgen.bcm.tmc.edu:9331/seq-search/struc-predict.html

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et al

.

Our next task was evaluating the similarity between thestructure of the central part of the

CG17604

-encoded pro-tein and the structure of central parts of the known proteinsof the transverse SC filaments in other organisms. Usingthe literature data [6, 16] on the width of the SC centralspace and the size of the coiled-coil parts of the Zip1protein with internal deletions and duplications of var-ious length (Fig. 2), we assessed the correlationbetween these parameters (

r

= 0.97;

P

< 0.001). In thissystem, we introduced the following data on

Drosophila

:the SC width (109 nm) [10] and the number of aminoacids determined using the ISREC procedure (531 aa) inthe central domain of the

CG17604

product. The pointthus obtained falls into the 95% confidence interval ofthe SC central space width in the examined organisms(Fig. 2). Thus, with respect to the coiled-coil part the

CG17604

product meets the requirements posed by theobserved SC central space width in

Drosophila.

According to the data on amino-acid homology [3], thepredicted product of the

CG17604

gene possess homol-ogy with a variety of proteins, many of which can formcoiled coils, such as the heavy myosin chain (

D. melano-gaster, Caenorhabditis elegans, Arabidopsis thaliana,

Dugesia japonica, Homo sapiens

), laminar proteins(

Mus musculus

) and the polar body protein NUF1(

S. cerevisiae

). This homology was shown for proteinsZip1 in yeast (myosin, filament proteins [8]), SCP1 inrat (proteins of the nuclear lamina, nuclear matrix,heavy myosin chain [9]), SCP1 in mouse (homologywith nuclear matrix proteins NUF1 of yeast andNUMA of mammals and with proteins of nuclear lam-ina [6]). It is interesting that by its amino-acid sequencethe product of the

CG17604

gene is homologous neitherto SCP1/SYN1 nor Zip1, and Zip1 is not homologous tothe SC central space proteins in mammals [8]. Thus, the

CG17604

gene is similar to the known genes forSC central space proteins also in this parameter.

For additional verification of the result obtained, wehave analyzed 49 meiotic mutations in

Drosophila

listed in FlyBase and selected those changing the fre-quency of meiotic recombination and thus capable toaffect the SC formation. We have examined 12 regionsof localization of these mutations and checked over200 predicted genes. The predicted protein products700–1000 aa in size were tested for the ability to forma coiled coil. Since none of these 200 virtual proteinscould form the rod-shaped protein structure of therequired length, none of them could be a component ofat least SC central space.

It is known that the isoelectric point (pI) of thewhole molecule or its domains is an important charac-teristic of proteins that participate in the DNA structureformation (probably including the SC proteins) [17].Using the ProtParam procedure, we have estimated pIof known SC central space proteins in various organ-isms and the

CG17604

product (Table 2). Note the sim-ilarity of the pI values for the C-terminal domain of allthese proteins (9.7–10.1). This domain is basic andassumed to participate in the interaction with DNA [9].The pI values of the total molecule and its coiled-coilpart are also similar. The specific feature of the

CG17604

gene product is the basic character of itsN-terminal domain (pI = 10), which sharply distin-guishes it from other proteins of this family, whoseN-terminal domains are acidic (pI ~ 4.2–5.9). Theobserved difference of the N-terminal domain formingthe SC central element may determine the specific mor-phology of the SC central element in

Drosophila

, i.e.,distinct striation and large width (about 32 nm) [10].

200

100

SC central space width

Number of amino acids

160140

100

180

120

80604020

500 700 1100 1300300 900

in coiled-coil region of protein molecule

Fig. 2.

The relationship between the SC central space widthand size of the coiled-coil region of the Zip1 protein mole-cule (after Tung and Roeder [7]). Designations as in Fig. 1.The open circle show the parameters of

Drosophila

SC andthe coiled-coil part of the predicted

CG17604

gene product.

Table 2.

Isoelectric points (pI) of SC central space proteins and their domains

Protein N-terminus Coiled-coil region C-terminus Total molecule

CG17604 10 4.93 9.72 5.91

Zipl 4.8 6.06 10.1 6.4

SCP1HS 5.86 5.31 9.73 5.84

SCP1MM 5.02 5.38 9.72 5.69

SCP1RN 4.2 5.33 9.79 5.56

Note: HS,

homo sapiens

; MM,

Mus musculus

; RN,

Rattus norvegicus

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Thus, we have found in the 88E6-89B2 region ofchromosome 3R in

Drosophila

only one gene whosevirtual protein is in many ways similar to the known SCproteins in other organisms. According to FlyBase, the

CG17604

gene is located in the 89A7-8 region. A num-ber of meiotic and close to meiotic genes whose nucle-otide sequences and corresponding gene products havenot been yet identified are located in the vicinity of

CG17604

. These genes include

c(3)G, rec

, and

tbi

(

fs160

). Using fine deletion mapping and recombina-tion analysis it was established that genes c(3)G andrec, according to different sources, are closely linkedand localized in the 89A2-5 region, whereas tbi wasmapped to the 89A4-5 region [15]. In addition, a geneof female sterility hls (also referred to as fs167 or anallele of spn; nucleotide sequence determined) islocated in subsection 89A5. What are the main func-tions of these genes? Mutant c(3)G alleles totally orpartially arrest meiotic recombination and block the SCformation [2]. In rec mutants, crossover frequencies aredecreased several tenfold [15], but the SC is formed [3].The tbi mutation causes female sterility due to off-spring mortality at the embryonic, or, more rarely,pupal, stage. The hls mutation also leads to sterilityimpairing symmetry and polarity of the egg [15].Which of these genes is associated with CG17604?

If this indeed is a gene coding for an SC protein (thisassumption can be proven by molecular-biologicalexperiments), two hypotheses can be advanced.

First, the gene that we found may be a known mei-otic gene: c(3)G or rec. If it is c(3)G, the mutation willlead to either total absence of the SC central space pro-tein or its serious damage that impairs the SC forma-tion. If it is rec, the protein may have slight defects,which do not impair the SC structure, but recombina-tion is hindered. A question arises as to the discrepancybetween the localization of c(3)G (and rec), on the onehand, and CG17604, on the other. This discrepancy ispossible, first, because mapping of the predicted geneswas conducted via their association with known identi-fied genes, which are are not very numerous so far [18].Second, FlyBase and NCBI give different localizationof the above genes. According to NCBI, CG17604 islocalized in subsection 89B17 rather than in region89A7-8 as in FlyBase.

The second hypothesis is as follows. The gene thatwe found is a structural SC gene while c(3)G and recare regulatory elements (enhancers or suppressors); allthese genes form a functional tandem. In any case,more experimental data are needed to draw further con-clusions. An indirect evidence for the attribution of theCG17604 gene product to the SC central space proteinfamily is the fact that none of the virtual protein prod-ucts of genes from the localization region of knownmeiotic mutations had the secondary structure charac-teristic of that family although the number of these pro-teins was high.

In our view, gene CG17604 is likely to be identicalto gene c(3)G encoding the SC central space protein inDrosophila. This suggestion can be tested experimentally.

After having submitted this paper, we becameacquainted with the Abstracts of the 17th EuropeanDrosophila Conference (September 1–5, 2001) whereHawley and coworkers [19] reported the experimentalisolation of the c(3)G gene product, which was structur-ally similar to the known SC proteins, Zip1 and SCP1.Using immunocytochemical analysis, these authorsshowed that this protein forms transverse filamentousstructures in bivalents at prophase I of oocytes. Thus,the assumption on the role of the C(3)G protein in chro-mosome synapsis has been proven experimentally.

ACKNOWLEDGMENTS

This work was supported by the Russian Foundationfor Basic Research (project no. 99-04-48 182).

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