Sex determination Drosophila Michael

and differentiation and Caenorhabditis McKeown

Salk Institute,

and Sanford San Diego,

in invertebrates: elegans

J. Madigan

California,

USA

Sex determination in flies and worms is controlled by cascades beginning with the X chromosome : autosome ratio and terminating in transcription factors. We are now gaining an understanding of the molecular mechanisms governing the largely post-transcriptional regulation of the intermediate steps in these cascades. Current

Opinion

in Cell Biology

1992, 4:94&954

Introduction

X chromosome counting Sex-lethal gene

Both Drosqbbila and Gxnorbabditis elegans use chromosomal sex determination systems in which the primary determinant is the X chromosome : autosome ratio (X : A ratio>. Such systems generate a requirement for both dosage compensation and sex regulation. In addition, germline sex differentiation and somatic sex diRerentiation are controlled by overlapping but not identical systems. We focus on the control of somatic sex differentiation, and the reader should note that the substantial complexity in analysis generated by the other systems is beiig ignored.

The functional state of Sex-lethal (Sxl) is set by the X : A ratio at the blastoderm stage of embryogenesis. Setting the activity of Sxl involves activation of a female-specific, embryo-specific Sxl early promoter (P,; Fig. 2) [7,8-•,9]. Determination of the X : A ratio is accomplished by the interaction of X-linked ‘numerator elements’ and autosomal ‘denominator elements’. The numerator elements are expressed in a transient, non-dosage compensated way at about the blastoderm stage such that female embryos (XX) produce twice as much product as male embryos (XY). At least three genes act as numerator elements: sisterless-a (s&-a>, sz3terless-b (sis-6) [lO,ll] and runt [ 12.1. Whereas s&a and s&-b act throughout the embryo, runt is spatially restricted [ 12.1. The sis-bgene encodes a basic helix-loop-helix (HLH) protein and corresponds to the T4 transcription unit of the acbaetescute complex, a gene involved in neurogenesis [7,10,11,13,14]. The runt gene encodes a nuclear protein that does not contain homology to any previously characterized DNA-binding or dimerization motifs. The evidence suggests that runt increases the ability of other numerator elements to activate Sxf [ 12.1. In addition to these numerator elements the maternally supplied daughterless (da) gene product, another basic HLH protein involved in neurogenesis, is required for proper transduction of information about the X:A ratio to Sxl[15,16]. As the basic HLH proteins Sis-b and Da are necessary for activating Sxl, it has been postulated that denominator elements may be inhibitory HLH proteins that interfere with the action of Sis-b and/or Da. Consistent with this hypothesis, ectopic expression of the product of the hairy gene, a negative HLH protein, prevents female-specific Sxl expression [ 171. The deaa$an gene, encoding a negatively acting HLH protein that is related to Hairy and is involved in neurogenesis, has been identified as an autosomal counting element. It is apparently neces-

Aside from the general considerations above, it is clear that Ilies and worms use substantially diRerent systems to control sex differentiation. This is in line with multiple observations that sex determination mechanisms vary substantially between species, even within the same phylum. This review concentrates on recent work, and there are a number of reviews that supply excellent background information [l-6].

Drosophila The basic outline of sex differentiation in Drosophila is shown in Fig. 1. Both genetic and molecular data suggest that regulation is a female-specific event. In the absence of female-specific regulation, male differentiation occurs. Female-specific regulation is the result of a series of positive regulators that connect an X : A ratio of 1 .O to female-specific regulation of the doublesex and amb&ex genes.

and activation

Abbreviations da-daughterless; X:A

HI&-helix-loop-helix; sis-sisterless; Sxl-Sex lethal; ratio-ratio of X chromosomes to autosomes; yryolk

@ Current

Biology

Ltd MN

0955-0674

tra-transformer; protein.

of the

Sex determination

and differentiation

in invertebrates

McKeown and Madigan

(a)

Present (b)

1 : 1

Present

1:2

Absent

->

Female

Female form

Female

Male form?

Male

Male form

Male

Present

Present (cl

Female form?

Absent

Positive

regulation

1-1

Negative

regulation

Fig. 1. The Drosophila

sex differentiation cascade. (a) The primary signal for sex determination is the X chromosome : autosome ratio (X : A). Regulation is entirely cell autonomous: each cell decides its sex independently of its neighbors. X chromosome-encoded positive factors (Sisterless ‘Siska, Sis-b and Runt) interact with autosomally encoded negative factors (Deadpan; Dpn) and maternally supplied factors (Daughterless and Extramacrochaete, not shown), to determine if Sex-lethal 6x0 is activated. If activated, Sxl positively regulates its own later expression (independent of the initial factors) as well as the activity of transformer kra). It also regulates dosage compensation and germline sex differentiation, which are beyond the scope of this review. If tra is active, its product and the product of the constitutively expressed Lra-2 gene regulate the doublesex (dsx) and ambisex genes, leading to female differentiation. dsxF requires the intersex (ix) gene product for function. If Sxl is not activated, as in males, tra RNA is expressed in an inactive form. If tra is inactive, then dsx and ambisex are expressed in their constitutive male modes of expression, leading to male differentiation. (b) The female and Cc) the male states of the hierarchy in terms of the protein products of the genes. Reviewed in II,61

sary for inhibition of Sxl activation in males [ 18**]. In addition, extrarnacrochaete (Emc), yet another negative HLH protein involved in neurogenesis, is required maternally for inactivation of S.v[ in males. Thus, the X :A ratio is transduced by the relative concentrations of maternally supplied du and emc gene products, X-encoded positive factors such as s&a, s&b and ruztt products and autosomal negative factors such as deaaan products. Many of these factors are HLH proteins known to interact with each other in development of the nervous system, so it is plausible that counting is mediated entirely by protein-protein interactions and not by DNA titration of factors. Assay of the X :A ratio occurs only once. Memory of the results of this assay is dependent on the presence or absence of Sxl protein and its effects on processing of Sxf transcripts from a distinct (late) promoter that is used in both males and females (Fig. 2) [9,19,20]. In the absence of Sxl protein (e.g. in males), Sxf RNAs contain an exon with several in-frame translation termination signals. In the presence of Sxl protein (e.g. in females) this exon is excluded; this produces RNAs that contain a long open reading frame that encodes functional Sxl proteins 119,201. As removal of the male exon requires Sxl, an autoregulatory feedback loop is established, in which the presence of Sxl protein leads to continued Sxl protein ex-

pression and lack of Sxl protein leads to continued lack of Sxl protein. This regulation of RNA processing by the C-1 gene product correlates with the fact that Sxf encodes a protein with two RNA-binding domain motifs [19]. Two additional gene products have been implicated in some aspects of Sxf regulation. The maternally supplied protein from the slzf(also known as fiz or Js(ZJ41621) gene is involved in some as yet poorly understood aspect of early Sxl regulation [ 21,221. The j7C2jd locus, a locus necessary for viability of both males and females, behaves as if it may be involved in efficient production of the late female-specific RNAs [ 231. In addition to the sex-specific splicing of Sxl RNAs there are a number of alternative splices that occur in both sexes and code for overlapping proteins with the same RNA-binding domains [ 241. Although these may be functionally different, at least one of them is capable of partial activity in all Sxl functions [20] Regulation

by Sex-lethal

of the transformer

gene

The tra gene gives rise to two classes of mRNA (Fig. 2). One is non-sex-specific and non-functional, while the other is both female-specific and functional [ 25,261. The female-specific transcript arises, in the presence of Sxl protein, through alternative 3’ splice site choice [ 27-291.

949

950

Cell differentiation

(a)

5’ P,

&-k

I

I

I

-3’

I

I

I

e

Female

Sx/

RNA

AUG

3

45

Male

Sxl

RNA

Fig.

2 Schematic

models

of

the

reg-

ulated RNAs involved in Drosophila sex differentiation. Different sections are drawn to different scales. Exons are rep-

2

resented introns

45 m = = e

Female

Sx/

RNA

AUC

as horizontal are thin lines.

translation initiation sites and UCA or UAG indicate translation termination sites. , the 3’ splice sites that are likely to be involved uiation. (a) Sex-lethal Pt is a female-specific,

fb) 1

2

promoter

3 Non-specific

AUG

lra

RNA

UAG

Female

AUG

rra

at exons represented

RNA

transcripts.

in

++A;-l’r+ UAG

Male

dsx

RNAs

2, 3 and with

AUG

UGA

dsx

RNA

of the doublesex

gene

The doublesex (a%~) gene is bifunctional, encoding sexspecific proteins identical for the first 397 amino acids,

5 are double

schematically lines. Addi-

(b) Processing

of transformer

and female-speonly in the splice of exon 2. The stop codons it, thus block-

the start codon in of doublesex (dsx)

male-specific (4) terminal

(5.6) exons.

or femaleThis gives

rise to alternative with common and sex-specific

dsx-encoded proteins amino-terminal regions carboxyl termini. ‘This

region contains ate 13 nucleotide

six copies repeat

has

The alternative processing occurs through Sxl blockage of the non-sex specific 3’ splice site [28,29]. Sxl and h-a share a common sequence at the 3’ ends of their regulated introns. This sequence, TTITTTTC, is necessary for regulation of tra splicing and for binding of Sxl protein to tra RNA ( [ 28,291; D Davis, M McKeown, unpublished data). This sequence is insufficient by itself for Sxl regulation (BA Sosnowski, RT Boggs, M McKeown, unpublished data). Sequences flanking the regulated splice site show little divergence across five Drosophila species [30*] and may be required for proper regulation of tra by Sxl.

about 4 h in desplicing events

site they use at the start non-specific RNA contains in the region unique to

different specific Female

AUG

of

ratio. Pt which

RNAs. The dsx RNAs contain three common exons (I-3) that are spliced to two

UAG

+++

activation

the X: A promoter,

on after Alternative

ing translation from exon 1. (c) Processing

1

,1”‘“-u+7lA

initial

to

RNAs. The non-specific cific tra RNAs differ

AUG

in gene autoreg(SxfI RNA patterns. embryo-specific

tional 3’ alternative processing events are not shown, but are indicated by the broken lines at the 3’ end of the

UCA

(cl

Regulation

used

Sxl in response is a non-sex-specific is switched velopment.

GAVe+

thickened lines; AUC indicates

been

implicated

of a degenersequence that

in regulation.

but with unique carboxyl termini (30 amino acids in females, 152 in males) [31]. These are derived from sex-specific RNAS resulting from alternative 3’ exon usage (Fig. 2). Default processing occurs in males. In the presence of the female-specific Tra protein and the constitutively expressed Tra-2 protein, female processing is induced [26,27,32*-34.1. This regulation is the result of activation of the female-specific 3’ splice site and is dependent upon a 13 nucleotide sequence repeated in degenerate form six times in the female exon [31,32*-34*,35]. These repeats are the binding site for Tra-2 protein [34*,36*], which contains a single RNAbinding domain [34*,35,37,38]. In HeLa cell extracts either Tra or Tra-2 proteins are capable of binding to the repeats, and each one is capable of inducing female type splicing by itself [36*]. These results are contrary to the in uivo observations as neither Tra nor Tra-2 proteins alone are sufficient for regulation in viuo [ 26,271.

Sex determination Regulation

of downstream

functions

by doublesex

Genetic data show that male and female Dsx proteins function to repress at least certain aspects of differentiation of the opposite sex. In the case of Dsx female this also requires the function of the intersexgene 1391. The yolk protein genes (vpl and ~$121 are expressed sex-specifically in the adult fat bodies under continuous control by the sex differentiation hierarchy. The fat body enhancer, a 127 base pair stage-, tissue-, and sex-specific enhancer of ~/PI and yp2, binds both the DsxM and DsxF proteins in vitro, presumably through the 397 amino acids they share at their amino-termim [40**]. Thus the RNA processing cascade ends in a gene that gives rise to sex-specific DNA-binding proteins. Burtis et al. [40*-l suggest two possibilities for in vivo regulation by Dsx: first, both Dsx proteins bind to enhancers of target genes, but the DsxM protein interferes with the function of an activator protein bound to female enhancers and vice versa; and second, the interaction of DsxF with another protein (possibly intersex) alters its DNA-binding specificity so that binding occurs adjacent to male-specific genes interfering with their activation.

Branching

pathways

Recent genetic experiments suggest that at least one gene other than dsxfunctions in the regulation of sexual differentiation under the control of tra and h-a-2 [41*-l. This additional gene(s) is inferred to regulate the differentiation of a pair of muscles found in the fifth abdominal segment of male flies. Development of these muscles is dependent on the sex of the nerves which enervate them [42]. Proper sex-specific differentiation of these muscles is dependent on the function of tra and tra-2 but is independent of the function of & The simplest explanation of these results is that there is a non-&gene controlled by tra and tru-2 that functions in the nervous system to control at least some aspects of nervous system sexual differentiation. The name ambkex has been suggested for this gene (B Taylor, personal communication).

Caenorhabditis

elegans

Wild type C eleguns exists as two sexes: self-fertilizing sequential hermaphrodites and males. The cascade controlling somatic sex differentiation in C e1egurr.scontains a series of negative regulatory steps that transmit information about the X : A ratio to the gene tra- I, which controls the expression of the male or hermaphrodite sexual phenotype (Fig. 3; reviewed in [ 2,5] >. At least four genes [X0 lethal (x01- 11, sdc-1, sdc-2 and sdc-31 function before the split between sex differentiation and dosage compensation, while at least seven (her- 1, tm-2, tru-3, fern-l, fern-? fern-3 and tra-2) are involved in sex but not dosage compensation. Genetic data show that maternal products for the genes sdc-1, tra-3, fern-l, fern-2 and fern-3 are supplied to embryos, suggesting that some regulation within the cascade is post-transcriptional. A temperature-sensitive allele of sdc-2 has its temperature-

and differentiation

in invertebrates

McKeown

and Madiaan

sensitive period during embryogenesis, showing that the hierarchy starts to act early in development and that saiz1 is involved in the establishment but not the maintenance of the determined state [43]. Thus genes downstream of s&-I must serve to remember the sexually determined state. Finally, mosaic analysis suggests that control of sex differentiation involves some extracellular interactions as s&-I and her-l mutants do not act cell autonomously [43,44**], and animals with intermediate X : A ratios (0.5 < X : A < 1.0) show unexpectedly high levels of correlation in sexual phenotype between adjacent cells derived from distinct lineages [44**]. This extracellular interaction must occur before the regulation of terminal differentiation as tru-I, the fhal gene in the pathway, acts in a cell-autonomous manner [45]. Recent molecular and molecular genetic experiments substantially clarify the mechanisms which underlie these genetic observations. RNA from the sdc-1 gene is expressed more strongly in embryos and adults than in larval stages, consistent with the observation that sdc-2 function is required only in embryos and has a strong maternal component [46]. The sdc-2 gene encodes a protein with seven TFRIA-like zinc fingers, suggesting that it functions by regulating nucleic acid metabolism, most probably transcription [ 461. This suggestion is consistent with the recent observation that her- 1 RNA expression is strongly or completely biased to ward X0 animals at all stages [47*]. This sex-specific bias is dependent on the function of all three sdc genes (DeLong, Plenefisch, Klein, and Meyer, referenced in [461; [47*] >. These results do not explain the apparent nonautonomy of sdc-I mutations. This may be because the calculation of the X : A ratio and stable activation or repression of downstream genes occurs before the cells that have lost the sdc-2 gene become depleted of maternal or zygotic product (see [43] for a more complete discussion). These results also fail to explain the fact that her-2 expression remains sex-specific even after the penod when sdc-1 is required, although they do suggest that there must be a stable repression of her-l after negative regulation by sdc-1. The her-1 gene encodes a low molecular weight secreted protein, in line with its non-ceil autonomous effects (M Perry and WB Wood quoted in [5]). The next downstream gene, tru-2, encodes a protein with multiple membrane-spanning domains. RNA encoding this protein is expressed in both sexes. The simplest model consistent with these data is that extracellular Her protein binds to Tra-2 membrane protein and inhibits its activity. From the genetics we infer that Tra-2 represses the activity of the fern genes or their products. The mechanism by which Her-l inhibits Tra-2 is not clear. Two of the potential target genes for h-a-2 activity (fernI and fern-j) have been cloned and sequenced [48,49]. Both encode soluble proteins and are expressed in males and hermaphrodites. RNA encoding the Fern-3 protein is supplied to both XX and X0 embryos in equal quantities, but it has an effect on somatic differentiation in males only, implying a post-transcriptional regulation of fern-3 activity. Recent studies show that this maternal RNA

951

952

Cell differentiation

Dosage

(al.

compensation

X:A sdc

fb)

1:2

(c)

1:l

,

3

fern-3

High

Low

High

Low

High

Low

Low

High

Low

High

Low

High

I=>

Positive

regulation

+I

Negative

CT

regulation

Fig. 3. The Caenorhabditis elegans somatic sex differentiation cascade. (a) The primary signal is the X chromosome : autosome ratio (X : A). After that, regulation involves a cascade of negative regulatory events. For example, in XX animals, xol activity is repressed. When xol activity is low, sdc activity is high. When sdc activity is high, her-7 is repressed. When her-? is low, k-2 activity is high. When (m-2 activity is high, activity of the fern genes is repressed. When /em activity is low, tra-l is active, leading to hermaphrodite differentiation and repression of male differentiation. Many of these genes are involved in a slightly different cascade controlling germline sex, which is not shown. At least some of the events controlling sex differentiation involve cell-cell interactions, so the sex of an individual cell deoends on its neiehbors. (b) The male and Cc) hermaohrodite states of activity for the various genes of the cascade. Reviewed in 1251. Fo; references to s&-3, see 1461.

is rapidly degraded in both sexes and is replaced by equal levels of zygotic RNA in both sexes, again suggesting post-transcriptional regulation [48]. In adults, hermaphrodite fern-.? RNA is restricted to the gennline (presumably to supply embryos). An equal level of fern3 RNA is found in adult males, but it is not clear if this RNA is also localized to the germline or if there is somatic male-specific RNA in adults [48]. If the latter were true, it would fit with present models, but this is not predicted on the basis of existing evidence. The Fern-l protein contains six copies of a repeated protein motif known as the ANK repeat (reviewed in [49]), which is found on a number of proteins involved in protein-protein interactions [50]. In some situations, for example the interaction of IxB with NF-xB, these repeats are necessary for formation of protein complexes that inactivate transcriptional activators. Models for the regulation of Fern activity by Tra-2 invoke a sequestration of the product (RNA or protein) of at least one fern gene (all three are required) such that it cannot function. As the fern-3 RNAs contain a nearly perfect 17 base inverted repeat in their 5’-UTR, it has been hypothesized that regulation of fern-3 may be translational . This 5’ region is different from a sequence in the 3’-UTR, which is necessary for germline-specific repression of fern-3 activity. When the fern products are produced and active they serve to eliminate b-a-1 function. Recent data indicate that the level of h-a-l RNA is the same in both sexes [ 51**], suggesting that regulation is post-transcriptional, perhaps through interaction of the protein with the ANK repeats of Fern-l. This is analogous to the negative interaction between I-xB and NF-xB. The formal similarity is strengthened by the fact that h-a- I encodes structures, especially the presence of two or Iive zinc fingers, sug-

gesting function as a transcription factor [51**1. These alternative protein isoforms are derived from RNAs differing in poly (A) sites and splicing patterns such that the two finger protein, except for its last nine amino acids, is a carboxyl-terminally truncated version of the five finger tra- 2 gene product. The two tra- 1 RNAs are differentially regulated in development, but the significance of this is not clear. Of eight nonsense mutations analyzed, two are in the common region and have strong (apparently null) phenotypes. Six are in the region specific to the longer protein and have severe but not complete null phenotypes. This suggests that the longer protein is necessary for tra- I function and the shorter protein is not sufficient ISl**l. One unexpected interaction observed from the molecular data is that activation of tra-2 increases the level of tra-2 RNA [52]. As noted above, h-a-2 RNA is present in both sexes, but there is a 15.fold higher level in hermaphrodites than in males. If XX animals are converted into males by tra-2 mutations, the level of tra-2 RNA drops to the normal male level. If X0 animals are converted to females by loss of function j&?? mutants (through activation of tra-I), the level of tra-2 RNA increases to the level found in hermaphrodites. Finally, feminization of X0 animals by her-l mutations (leading to the loss of an extracellular signaling molecule) results in levels of tra-2 RNA comparable to that in wild type XX animals [52]. This last result is the only one consistent with the genetic pathway (lacking the feedback loop of Fig. 3) that was originally proposed. This is probably a reflection of the repression of tra-1 activity that occurs in her- I mutant animals and not of direct regulation of tra-2 RNA by her- 1. There are certain advantages to this system, with a mixture of extracellular signaling and intracellular feedback. Specifically, the system is buffered against the possibil-

Sex determination

ity of individual cells making incorrect sexual choices (leading to intersexual organismal development and potential sterility) [5,53-l. High levels of Tra-2 protein in hermaphrodite cells (in response to h-a-1 activity) make it unlikely that low-level expression of Her-l protein will be sufficient to inactivate Tra-2 in those cells. This reinforces the hermaphrodite developmental pathway. Low levels of Tra-2 in males (at least in part the result of lack of Tra-1 activation) render these cells particularly sensitive to inactivation of all the Tra-2 by Her-l protein, thus enforcing the male pathway.

and differentiation

between Two Alternative its 1980. 119:829-862. 12. .

McKeown and Madiaan

in invertebrates

Stable Expression States. Genet-

Segmentation Gene Numerator Element Necessary for the Uniform Activation of the Sex-determining Gene Sex-lethaL Genes Deu 1991. 5:2176-2157.

DLIFFY JB,

GERGEN JP: The

Drosophila

runt Acts as a Position-specific

Identification

of runf

as an additional

numerator

element.

13.

Torut!& M, SANCHEZ L: The scute (T4) Gene Acts as a Numerator Element of the X :A Signal that Determines the State of Activity of Sex-lethal in Drosophila L?MBO J 1989, 8:3079-3086.

14.

TORREZ

L The sisterlessb Function of the Gene scute is Restricted to the Stage when the X :A Ration Determines the Activity of Sex-IetbaL De cjelopmenf 1991, 113:715-722. M,

SANCHEZ

Drosophila

IS.

Acknowledgments

CALIDSM, VASSIN

H, BRAND M, TUMA R, JAN LY, JAN YN: duugb a Dtosopbilu Gene Essential for Both Neurogenesis

terless,

and Sex Determination, Has Sequence Similarities to myc and the acbeutescute Complex. Cell 1988, 55:1061-1067.

Work on sex differentiation in the authors’ laboratory is supported by a grant from the NIH fo MM. SJM is an NIH Postdoctoral Trainee. The authors are recipients of Cancer Center Core Grant CA.14195. We thank various colleagues for transmission of information about their most recent work and apologize for any subjecu; incompletely covered or left out due fo space considerations.

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necessary upstream

for regulation of the region

BURTIS K, BAKER BS: Drosophila doublesex Somatic Sexual Differentiation by Producing Spliced mRNAs Encoding Related Sex-specfic Cell 1989, 56:997-1010.

of Ira conserved

ly

Gene Controls Alternatively Polypeptides.

RYNER LC, BAK!ZR BS: Regulation of doublesex pre-mRNA Processing Occurs by 3’-Splice Site Activation. Gerles De13 1991, 5:2071-2085. This paper and [33*,34*] come to the important conclusions that rlsr regulation involves Tra and Tra-2 dependent activation of the &X female-specific 3’ splice site and rhar regulation depends on a set of six copies of a thirteen-nucleotide repeated sequence present in the female-specific exon. In addition, this paper seems to rule out positive regulation of female-specific polyadenylation.

32. .

HOSHIJIMA K, INOUE K. HICLICHI I, SAKAMOTO H. SHI~ILIRA Y: Control of doublesex Alternative Splicing by transfomer and transformer-2 in Drosophila Science 1991, 252:833-836. One of three papers [32*-34*] showing that regulation of &.x is via regulation of the activity of the female-specific 3’ splice site.

33. .

HEDIEY ML, mu\TIs T: Sex-specific Splicing and Polyadenylation of dsx pre-mRNA Requires a Sequence that Binds Specifically fo tra-2 Prorein in Vitro. Cell 1991, 65:57’+586. One of three papers [ 32*-34*] showing that regulation of &is via regulation of the activlry of the female-specific 3’ splice site. Also shows that Tra-2 protein binds fo the 13 nucleotide repeats of dsx Ryner and Baker 132.1 disagree with the conclusion that poly(A) addition is also a direct target of regulation.

fo a Sex-specilic Yolk 10:2577-2582. The first direct demonstration feet by controlling transcription 41. ..

36. .

NACOSHI RN, BAKER BS: Regulation of the Sex-specific RNA Splicing af the Drosophila doublesex Gene: cis-acting Mutations in Exon Sequences Alter Sex-specific RNA Splicing Patterns. Genes Dev 1990, 4~8997.

M, MANIATIS T: Positive Control of pre-mRNA Splicing 1992, 256:237-240. In vitro splicing of &x RNA in HeIa extracts supplemented with Tra or Tra-2 proteins. In this heterologous system, either Tra or Tra-2 proteins increase splicing to the dw female splice site, contrary to the in clivo situation. Also contains evidence that Tra protein can bind to rhe 13 nucleotide repeats of du: 37.

nAN

in Vitro. Science

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KC, COSCHIGANO KT, BAKER BS, WENSINK Proteins of Drosophila melanogaster

on in

the

BJ:

Gene

Enhancer.

thar Dsx protein of downstream

Differentiation

Drosophila

melanogaster Genes doublesex

EMEOJ

1991,

exerts its regulatov targets.

ef.

of a Male-specihc Muscle in Does not Require the Sex-deteror intersex. Getreticr 1992, 132:

mining in press. Exciting obsenation strongly suggesting the existence way involving regulation of sexual differentiation by

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tra

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VIUENELR’E Determination

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HLINTER CP. Wool WB: Evidence from Mosaic Analysis of the MascuUnhing Gene her-l for Cell Interactions in C. elegans Sex Determination. Nalrtre 1992, 355:551-555. The critical first gene in the pathway committed to sex differentiation exerts its effects on phenotype in a non-cell aufonomous manner. 45.

46.

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tra-1

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TREK C, PLIRNEU B. GAVINSKI S, HAGE~~AN J, C~L\~~RUN C, WOOD WB: Sex-specific Transcriptional Regulation of the C. elegans Sex-determining Gene ber.1. Mech Dev 1991. 34:43-55. Describes how the first gene committed fo sex differentiation is regulated by transcription; this correlates wiith the suggestion that the Sdc- 1 protein has a DNA-binding protein strucrure [46].

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AHRINGER J, ROSENQIUST TA, LOX’SON DN, KIMRII J: The Caenorbabditis elegans Sex Determining Gene fern-3 is Regulated Post Transcriptionally. fi\#O J 1992, 11:23032310.

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MICHAELY P. BENNY V: The Involved in Macromolecular 1992, 2:127-129.

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34. .

35.

TAYLOR

Protein

ANK Repeat: Recognition.

a Ubiquitous Morif Trendy Cell Biol

~XRKOWER D, HODGKIN J: Molecular Analysis of the C. elegans Sex-determination Gene tra- I: a Gene Encoding Two Zinc Finger Proteins. Cell 1992, 70~237-249. A strongly suggestive observation that the end gene in the sex cascade functions by controlling transcription. In addition it contains evidence that sex-specific regulation of this transcriptional regulator must be post-transcriptional. 51.

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53. ..

Action

Drosophila

PC: The DouBind Directly

M McKeown and SJ Madigan, Molecular tory, The Salk Institute, PO Box 85800, USA

Biology and Virology San Diego, California

Iabora92186,