Gene structure and organization in Caenorhabditis elegans

692

Gene structure and organization Thomas Blumenthal* The sequencing genome

-14 000 genes

One of its most interesting introns

and intergenic

surprisingly,

-25%

transcription

features

distances

of genes

elegans

- is -50%

complete.

is its compactness;

are unusually

are contained

units (operons)

with only -100

small and,

in polycistronic bp between

genes.

Addresses ‘Department

of Biology,

Indiana

University,

Bloomington,

Indiana

47405, USA; e-mail: [email protected] +Genome Sequencing Center, Washington University School of Medicine, St. Louis, Missouri e-mail: [email protected] Current

Opinion

0 Current

Biology

in Genetics Ltd ISSN

63106,

8 Development

6:692-696

0959-437X

Introduction The nematode Caenorhabditis elegarrs was chosen just over 20 years ago by Sydney Brenner as a model organism for the study of development and neurobiology [l]. It has now attracted a large number of researchers who focus on a wide variety of aspects of development, cellular architecture, and basic molecular biology. One of the major projects in recent years has been the physical mapping and sequencing of the entire 1OOhlb genome by two groups: the Genome Sequencing Center at the Washington University School of Rledicine in the USA and the Sanger Centre in the UK [2,3’,4]. The scheduled completion of the entire sequence of the C. elegans nuclear genome in mid-1998 will be the first for a complex, multicellular organism. As of October 1996, >SOhlb of genome sequence had been finished with approximately 25 hlb unfinished but in contigs of >l kb; by another this time, the genome was being sequenced at a rate of >Z.Shlb per month by the two groups. The completed sequence is publicly available through the World Wide Web [5,6]. This high rate of progress has been possible, to a large extent, because of the existence of a near-complete clonal physical map [3*] which allows the selection of minimally overlapping clones. The sequencing strategy is primarily a shotgun approach of sequencing random subclones derived from individual cosmids followed by a directed, or finishing, phase that closes gaps, resolves compressions and discrepancies, and compares sequences from different dye terminators [7-l. of the central

entire

X chromosome,

not just

because

of the

high

chromosomes (YACs) at present, but preliminary results indicate that some of this 20 Mb is represented in single copy cosmid libraries (S Chissoe, hI hlarra, A Coulson, R Shownkeen, personal communication). The remainder of the sequence is expected to come from subclones derived from YACs.

Prediction of genes from the raw sequence 1996,

expressed sequence tags yeast artificial chromosomes

The initial focus on the gene-rich

the

information content and importance of genes but also because these regions have deep cosmid coverage. Only -8Ohlb of the genome is represented in cosmids, the sequence of which should be completed by mid-1997: the remaining 20 Mb is represented only in yeast artificial

USA;

Abbreviations ESTs YACs

elegans

and John Spiethl

of the 100 Mb Caenorhabditis

- containing

in Caenorhabditis

sequencing effort has centered regions of the autosomes and

In general, gene prediction is a synthesis involving a computer program such as GENEFINDER (P Green, L Hillier, personal communication) plus other information. GENEFINDER uses statistical criteria-primarily log likelihood ratios-to identify likely genes within a region of genomic sequence. This automated approach uses criteria similar to the ones used to predict genes manually: coding regions have different characteristics from non-coding regions and they are separated from one another by recognizable splicing signals. In C. elegans. coding regions are significantly more G/C-rich than are non-coding regions (46% versus 30%) [S”]. It is possible to find open reading frames by connecting sequences that are relatively G/C-rich through consensus splice sites. In C. elegans, the consensus 5’ splice site is the same as in vertebrates but the 3’ splice site (UUUUCAG/R) is unusually long and highly conserved [S”]. This consensus is useful in enabling GENEFINDER to predict authentic genes. In addition, a large set of C. elegaas expressed sequence tags (EST& available in confirming GENEFINDER personal communication).

Characteristics

at [9]) has predictions

been useful (Y Kohara,

of the genome

The sequence studied to date has already provided some interesting predictions and insights into the organization of the genome. For instance, it was estimated that the -14000 genes in total, on the basis of genome conrdins the ratio of predicted genes to exactly matching cDNA sequences [lo”]. The organization of the X chromosome is significantly different from that of the autosomes. The density of predicted genes on X is lower than on the autosomes: 20% of the genome is coding on the X versus -30% on the autosomes [lo”]. This may reflect gene clustering near the center of the autosomes, whereas genes on the X appear to be distributed more uniformly, a feature first noticed in Brenner’s early studies of genes defined by mutation [l]. The proportion of genes with matching cDNAs is also smaller on X (23% on X, 35% on the autosomes) [Z], which could possibly be caused by the

Gene strudure

incorrect introns

prediction

of genes

and increased

spacing

on X resulting between

from

larger

genes.

T12A2.2

T12A2.1

TlZA2.9

z - 5000

1 8

I

(srg-8)

T12A2.10

(srg-9)

Cl 8F10.4

(srg-1)

Cl 8F10.5

(srg-2)

T12A2.12

(srg-4)

T12A2.1

1 (srg-5)

T12A2.13

(srg-6)

Cl 8F10.8

(srg-7)

Cl 8F10.6

(srg-3)

10000

C18FlO

in Caenorbabditis elegans Blumenthal

that one of the criteria for choosing small genome, but this compactness variety of interesting and surprising

and Spieth

693

this organism was its is manifested in a ways. These include

small introns and the close proximity of genes, including genes within genes (Fig. l), and operons of several kinds. In general, C. elegans genes are similar to those of other animals, comprising short exons interspersed with introns. Indeed, all C. elegans protein-coding genes-excluding those that encode most histoneshave introns and most have multiple introns.

Figure 1

T12A2

and organization

- 15000

Cl 8F10.2

C18F10.7

C. elegans exons have a modal distribution of -80-250 bp (Fig. Z), which is similar to vertebrate exons. C. elegans introns however, tend to be much shorter than those of vertebrates (Fig. 2); more than half are shorter than 60 bp, which is too short for splicing in vertebrates. The most common length is 48 bp and the shortest reported intron is only 30 bp long [ 111. Nevertheless, some C. elegans genes do contain long introns; for example, the first intron of utlr-7 is 18 kb long [12]. Furthermore, genes themselves can be quite long, having multiple exons and introns: urrc-22, for instance is >35 kb [13]. One important difference between C. elegans and other animals is that, in the latter, the promoter defines the 5’ end of the first exon, whereas in all nematodes-including C. e/egans-for a large proportion of the genes (-70% in C. elegans [14]), the promoter is at the 5’ end of the outron [15]. An outron is an intron-like sequence at the 5’ end of the pre-mRNA that is removed by trans-splicing (reviewed in [16,17]). Trans-splicing is closely related to intron removal but it replaces the outron with a 22 nucleotide leader, termed the spliced leader, which has practical consequences for the study of C. elegans genes. It is often difficult to pinpoint the location of the promoter, because trtznJ-splicing removes the outron efficiently and, therefore, the 5’ end of the pre-mRNA. The length of most outrons has thus not been determined, although the few that are known are in the neighborhood of 60-200 bp [P]. Hence, it is usually difficult to define the exact 5’ boundary of a gene that is subject to trans-splicing and, therefore, the length of the gene itself.

Kb c 1996 Current

Opjn~on inGenetics&Development

High gene density of the C. elegans genome. This is an ACeDB representation of the predicted genes in a region of overlap between

cosmrds

T12A2

and Cl 8FlO

on chromosome

3. The open

rectangles and connecting lines represent exons and introns of predrcted genes respectively with those to the left of the vertical bar on one strand and those to right on the other. The scale bar is in kb with 0 at the end of Cl 8FlO. There are nine seven-transmembrane receptors (srg-1 through srg-9) [26”] that fall completely within an intron of a gene on the other strand, T12A2.1.

A compact genome One of the most striking is its compactness. This

features of the C. &guns genome is not surprising if one considers

The Caenorhabditis operons

elegans

genome

contains

The existence of trans-splicing in C. elegam has permitted the development of another unusual feature: polycistronic transcription units similar to bacterial operons ([18]; reviewed in [19]). About a quarter of all the genes are contained in these operons [14], which often contain just two genes but can be much larger: clusters of up to six genes have been observed and these are likely to represent single operons (e.g. cosmid CZ6E6; T Blumenthal, unpublished observations). A cosmid containing two typical C. elegans operons is depicted in Figure 3. The operons are transcribed from a single promoter upstream of the first gene but are processed into monocisrronic mRNAs by 3’ end formation and tmns-splicing. Each of the genes

694

Genomes and evolution

Figure 2 C. elegans distributions. number

(a) intron and (b) exon length Each bar represents

of introns

class. The survey included and 862

the

(a)

or exons in each size 669

introns

400

exons. The inset in (a) shows

an expanded

plot of the small introns.

Each bar represents introns of a specific length: O-20, 21-40, 41-60, and so on. See [8**1 for details.

Length (bp) (b) 100

25

in the operon has a 3’ end formation signal which results in cleavage and polyadenylation end of the RNA. In addition, the next gene is processed by tmrzs-splicing to form its 5’ is some evidence that these two processes

(AAUAAA) at the 3’ downstream end. There are coupled

mechanistically ([18]; S Kuersten eta/., unpublished data). In general, the distance between the 3’ end of an upstream gene and the 5’ end of a downstream gene in an operon is -100 bp, although distances of up to 400 bp have been observed [S”]. There is a specialized spliced leader, called SL2 [20], used exclusively at internal tmns-splice sites in polycistronic pre-mRNAs. The presence of this leader at the 5’ end of an mRNA is taken as the definitive

indication that indeed represent

a cluster of closely an operon [19].

spaced

genes

does

.4 second, rarer class of operon also exists in the C. elegclw genome. In this class, there are no bases between the genes: tmns-splicing (using the more common SLl in this case) accomplishes both 3’ and 5’ end formation ([Zl]; I Korf and S Strome. personal communication; T Blumenthal and L Xu, unpublished data). Polyadenylation occurs at the free 3’ end of the upstream gene, created by tralrs-splicing of the downstream gene. The existence of both these classes of operons complicates the task of gene prediction by programs such as GENEFINDER,

Gene

which

has sometimes

combined

separate

structure

genes

because

and organization

in Caenorhabditis elegans Blumenthal and Spieth

In bacteria,

operons

provide

a mechanism

695

for the co-reg-

of their close proximity to each other. These ‘miscalls’ can often be resolved by finding homology to genes from other organisms or by C. elegans cDNAs, especially those

ulation of genes the products of which function together. The cistrons are all contained on a single mRNA that is translated in sequence from the 5’ end. In the case of the

containing

C. elegans operons, the genes are translated from separate mRNAs formed from a single polycistronic pre-mRNA. Nevertheless, they are all transcribed from a single promoter at the 5’ end of the cluster and are thus expressed in the same cells at the same time. Hence, the operons could be a means of co-expression of functionally-related

Figure

an untranslated

region

sequence.

3

genes, as in bacteria. If so, this would have very important implications for other biologists, whether or not involved in C. e/egam. The discovery of a gene in an operon would therefore imply that the other genes contained in the same operon would function in the same process. One could thus rapidly expand the available molecular arsenal for studying a particular process by studying the other genes

9

‘;

ZU637.3

q

ZU637.4

in the same

ZK637.5 (ArsA)

ZK637.6

ZK637.7 (lin-9) ZK637

4

25 000

ZK637.6a and Eb (TJG/proton pump)

ZK637.9 - 30 000

F!l

ZK637.10 (Glutathlone

reductase)

,c

A cosmid containing two typical C. elegans operons. This is an ACeDB representation of the genes on ZK637, a cosmid from chromosome 3. The open rectangles and connecting lines represent exons and introns of predicted genes with those to the left of the vertical bar on one strand and those to right on the other. The scale bar is in kb with 0 at the top of ZK637. ZK637.3, ZK637.4 and ZK637.5 are in one operon, whereas ZK637.8, ZK637.9 and ZK637.10 are In another operon [14]. ZK637.8a and ZK637.8b are two alternatively spliced forms determined from cDNA clones.

operon.

As the C. elegans genome project reaches its conclusion, an increasing number of researchers working with other organisms are finding homologs of their genes of interest in C. elegarrs, and about a quarter of these genes are expected to be in operons. Can these researchers reasonably expect that the other genes in these operons are functionally related to the one they have been studying? Unfortunately, it is too soon to tell for certain. In most cases, operons contain genes the functions of which have not yet been determined. In other cases, operons appear to contain genes the products of which might be expressed ubiquitously and thus not regulated at all. There are a few clear cases, however, of functionally related genes in C. elegans operons. For example, the /in-ls’a and b genes encode products that are unrelated to each other by sequence but which function together in vulva] determination [Z&23]. A second example is the deg-3 operon [24]; deg.3 encodes an acetylcholine receptor subunit and is co-transcribed in an operon with a gene that likely encodes another subunit of the same receptor (hl Treinin, hl Chalfie, persona1 communication). A third operon contains two genes the products of which function sequentially in the post-translational processing of collagen (A Page, personal communication). Thus, there are several clear examples of operons that appear to function in co-regulating related genes. Although we cannot yet conclude that the presence of two genes in the same operon constitutesprrnla jzcie evidence that they are related functionally, there are now enough examples to make it seem wise to investigate that possibility routinely.

Uses of the sequence Though the genome sequence is incomplete, the “postsequence genetics” of C. elegam [25*] has already started to take shape, with the existing sequence already having been put to some fruitful and interesting uses. For example, in an attempt to answer the question of how

696

Genomes and evolution

a small number (32) of chemosensory neurons generates responses to a much larger number of chemicals, Troemel et a/. [26”] have identified >40 members of a divergent

Figure 4

q f=

MM14H7.7

family of G protein-coupled receptors that may be chemosensory receptors. The authors have also shown that 11 of the 14 genes tested were expressed in subsets of chemosensory neurons with a single neuron potentially expressing at least four different receptor genes; they thus appear to have identified olfactory receptors in C. elegans that had escaped detection by homology searches with vertebrate olfactory receptors.

- 5000

Other steps toward the functional characterization of genes predicted from the sequence have been taken by the visualization of their expression patterns using either IacZ fusions in transformed lines [27’] or high resolution fluorescence h &IL hybridization in whole worms [W]. Not only will these studies be valuable in the interpretation of genome sequence data by providing the tissue, cell-type and developmental expression pattern of predicted genes-images are now being included in ACeDB, an object-oriented database [ZsL] - but they will also be another test of the accuracy of the sequence data and gene predictions.

- 10000

@

-

MM1 4H7.6

15000

MM14H7 0

52

- 20000

MM1 4H7.8

w

- 25000

=a

MM1 4H7.4

- 30000

-El a se

MM14H7.3 MM1 4H7.9

MM1 4H7.2

- 35000 EI EI

MM14H7.1

I Kb C. briggsae

cosmid

MM14H7

C’ 1996 Current Opln~on tn Genetics & DevelopmentJ showing

similarity

to C. elegans

in

coding regions. This is an ACeDB representation of the genes on MM1 4H7, a cosmid from chromosome 3. The open rectangles and connecting lines represent exons and introns of predicted genes with those to the left of the vertical bar on one strand and those to right on the other. The thicker filled rectangles are representations of the Blastn scores [34] (where width of the rectangles indicates the size of the score) of homologous genes on C. elegans cosmid T20G5. Scale bar is in kb with 0 at the top of MM1 4H7.

Evolutionary biology has also benefited from the C. e/egam genome sequence, as was shown recently by the identification of a T-box gene family [30]. These are genes with a region of homology to the mouse T locus, which is known to play an important and conserved role in vertebrate development. Their existence in C. elegnns demonstrates the ancient nature of this gene family and leads one to wonder what role they might play in nematode development. Phylogenetic comparisons of sequences between related species-such as between Caenor-hnbdikr eiepns and its relative Caenorhabditiis br&saehave proven a powerful tool in identifying conserved elements important for function and regulation (e.g. 131,321). A comparison of cosmid-size regions of C. elegams and cl’. hl@JNe is underway at present as part of the c’. e/<~~~rs Genome Project (M Marra, personal communication). Figure 4 shows such a comparison in ACeDB (J Couch, personal communication). The conservation of coding and potential regulatory regions between the two species will be useful in predicting genes. Others have expanded the utility of phylogenetic comparisons by taking advantage of the synteny and conservation of gent linkage between the two species to isolate homologs that have been difficult to identify by hybridization [33].

Conclusions The availability of the complete sequence of the genome of a metazoan organism, the nematode worm C. e/pRcnz.s,will be an important milestone in the progress of molecular genetics. It will enable us to view, for the first time, the entire array of genes required to build a complex,

Gene structure and organization in Caenorhabditis elegans Blumenthal

multicellular

animal.

In

addition,

it will yield

important

insight into how selection operates at the level of the genome. Already, the C. elegans genome sequence has provided surprising examples of gene arrangements-such as genes within genes and clusters of genes in operons. In addition, the genome sequence has revealed numerous examples of families had previously been

of genes present in C. elegam which known only in ocher organisms, thus

providing evidence for the fundamental nature of the functions that their products perform. Finally, the C. elegans Genome Project is providing numerous homologs of genes being studied by workers investigating vertebrate and other organisms. Comparison of the sequence of these homologs with their counterparts from other organisms can provide insight into the evolution and function of the genes and processes under study. The vast amount of new sequence being submitted at present to the databases represents a challenge to the C. elegans field, as well as other researchers, to devise novel ways of exploiting this resource. The possibility of focusing on the products of all the genes to learn what processes they participate in would open entirely new avenues for research. New kinds of research projects are becoming possible and only await creative minds to initiate them.

References

and recommended

Papers of particular interest, published have been highhghted as: . l

*

reading

697

and Spieth

8.

Blumenthal T, Steward K: RNA processing and gene structure. In C. elegans II. Edited by Riddle D, Blumenthal T, Meyer B, Priess J. Cold Spring Harbor, New York: Cold Spring_ Harbor Laboratorv Press; 1996:117-l 45. This chapter in another new book devoted to the bioloov ~.z “1 of C. eleoans summarizes the current state of knowledge of C. elegans gene structure and pre-mRNA processing. It includes a new survey of characteristics of C. elegans genes and splice sites. ..

9.

DNA Data Bank of Japan (DDBJ): C. elegans Database Links on World Wide Web URL: http://www.ddbj.nig.ac.jp/ htmIs/c-elegans/html/CE_INDEX.htm

10. ..

Waterston RH, Sulston JE, Coulson AR: The genome. In C. elegans II. Edited by Riddle D, Blumenthal T, Meyer B, Priess J. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1996:23-45. This chapter summarizes the current state of the C. elegans genome sequencing project. It contains the most up-to-date attempt to identify general characteristics of the genome. 11.

Sibley MH, Johnson JJ, Mello CC, Kramer JM: Genetic identification, sequence, and alternative splicing of the Caenorhabditis elegans alpha 2 (IV) collagen gene. J Cell Biol 1993, 123:255-264.

12.

Starich TA, Herman RK, Shaw JE: Molecular and genetic analysis of unc-7, a Caenorhabditis elegans gene required for coordinated locomotion. Generics 1993, 133:527-541,

13.

Benian GM, CHernault SW, Morris ME: Additional sequence complexity in the muscle gene, unc-22, and its encoded protein, twitchin, of Caenorhabditis elegans. Genetics 1993, 134:1097-l 104.

14.

Zorio DAR, Cheng NN, Blumenthal T, Spieth J: Operons represent a common form of chromosomal organization elegans. Nature 1994, 372:270-272.

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Spieth J, Brooke G, Kuerston S, Lea K, Blumenthal T: Operons in C. elegans: polycistronic mRNA precursors are processed by trans-splicing of SL2 to downstream coding regions. Cell 1993, 73~521-532.

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Hodgkin J, Plasterk RHA, Waterston R: The nematode Caenorhabditis efegans and its genome. Science 1995, 270:41 O-41 4.

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Favello A, Hillier L, Wilson RK: Genomic DNA sequencing methods. In Methods in Cell Biology. Caenorhabditis elegans: Modern Biological Analysis of an Organism. Edited by Epstetn HF, Shakes DC. San Diego: Academic Press, Inc.; 1995, 46:551-569. As with [3’]. another chapter in the new book of methods used by C. elegans researchers, this one describing the strategy being used to produce the C. elegans genome sequence.

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26. .

Birchall PS, Fishpool RM, Albertson DG: Expression patterns of predicted genes from the C. elegans genome sequence visualized by FISH in whole organisms. Nat Genet 1995, II:31 4-320. This paper reports development of a technique that uses flourescently labeled probes and confocal imaging to sample expression patterns of genes predicted by the genome project. This approach is complementary to the approach of analyzing /acZ fusions described in [27’). 29. .

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