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Female sperm storage is a key factor for reproductive success in a variety of organisms, including Drosophila melanogaster. The spermathecae, one of the Drosophila sperm storage organs, has been suggested as a long-term storage organ because its secreted substances may enhance the quality of sperm storage. Glucose dehydrogenase (GLD) is widely expressed and secreted in the spermathecal ducts among species of the genus Drosophila. This highly conserved expression pattern suggests that this enzyme might have an important role in female fertility. Here, we examine the function of GLD in sperm storage and utilization using Gld-null mutant females. The absence of GLD reduced the amount of sperm stored in the spermathecae and led to a highly asymmetrical sperm distribution in the two spermathecal capsules of the mutant females. The storage defect was especially severe when the mutant females were crossed to a Gld-mutant male that had previously mated a few hours before the experimental cross. Under this mating condition, the mutant females stored in the spermathecae only one-third of the sperm amount of the wild-type control females. In addition, the mutant females used stored sperm at a slower rate over a longer period compared with wild-type females. Thus, our results indicate that GLD facilitates both sperm uptake and release through the spermathecal ducts.

Humans afflicted with the Wolcott-Rallison syndrome and mice deficient for PERK (pancreatic endoplasmic reticulum eIF2alpha kinase) show severe postnatal growth retardation. In mice, growth retardation in Perk-/- mutants is manifested within the first few days of neonatal development. Growth parameters of Perk-/- mice, including comparison of body weight to length and organ weights, are consistent with proportional dwarfism. Tibia growth plates exhibited a reduction in proliferative and hypertrophic chondrocytes underlying the longitudinal growth retardation. Neonatal Perk-/- deficient mice show a 75% reduction in liver IGF-I mRNA and serum IGF-I within the first week, whereas the expression of IGF-I mRNA in most other tissues is normal. Injections of IGF-I partially reversed the growth retardation of the Perk-/- mice, whereas GH had no effect. Transgenic rescue of PERK activity in the insulin- secreting beta-cells of the Perk-/- mice reversed the juvenile but not the neonatal growth retardation. We provide evidence that circulating IGF-I is derived from neonatal liver but is independent of GH at this stage. We propose that PERK is required to regulate the expression of IGF-I in the liver during the neonatal period, when IGF-I expression is GH-independent, and that the lack of this regulation results in severe neonatal growth retardation.

Nuclear factor kappaB (NF-kappaB) serves to coordinate the transcription of genes in response to diverse environmental stresses. In this report we show that phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2) is fundamental to the process by which many stress signals activate NF-kappaB. Phosphorylation of this translation factor is carried out by a family of protein kinases that each respond to distinct stress conditions. During impaired protein folding and assembly in the endoplasmic reticulum (ER), phosphorylation of eIF2alpha by PEK (Perk or EIF2AK3) is essential for induction of NF-kappaB transcriptional activity. The mechanism by which NF-kappaB is activated during ER stress entails the release, but not the degradation, of the inhibitory protein IkappaB. During amino acid deprivation, phosphorylation of eIF2alpha by GCN2 (EIF2AK4) signals the activation of NF-kappaB. Furthermore, inhibition of general translation or transcription by cycloheximide and actinomycin D, respectively, elicits the eIF2alpha phosphorylation required for induction of NF-kappaB. Together, these studies suggest that eIF2alpha kinases monitor and are activated by a range of stress conditions that affect transcription and protein synthesis and assembly, and the resulting eIFalpha phosphorylation is central to activation of the NF-kappaB. The absence of NF-kappaB-mediated transcription and its antiapoptotic function provides an explanation for why eIF2alpha kinase deficiency in diseases such as Wolcott-Rallison syndrome leads to cellular apoptosis and disease.

The GCN2 eIF2alpha kinase is essential for activation of the general amino acid control pathway in yeast when one or more amino acids become limiting for growth. GCN2's function in mammals is unknown, but must differ, since mammals, unlike yeast, can synthesize only half of the standard 20 amino acids. To investigate the function of mammalian GCN2, we have generated a Gcn2(-/-) knockout strain of mice. Gcn2(-/-) mice are viable, fertile, and exhibit no phenotypic abnormalities under standard growth conditions. However, prenatal and neonatal mortalities are significantly increased in Gcn2(-/-) mice whose mothers were reared on leucine-, tryptophan-, or glycine-deficient diets during gestation. Leucine deprivation produced the most pronounced effect, with a 63% reduction in the expected number of viable neonatal mice. Cultured embryonic stem cells derived from Gcn2(-/-) mice failed to show the normal induction of eIF2alpha phosphorylation in cells deprived of leucine. To assess the biochemical effects of the loss of GCN2 in the whole animal, liver perfusion experiments were conducted. Histidine limitation in the presence of histidinol induced a twofold increase in the phosphorylation of eIF2alpha and a concomitant reduction in eIF2B activity in perfused livers from wild-type mice, but no changes in livers from Gcn2(-/-) mice.

Phosphorylation of eukaryotic initiation factor 2 alpha (eIF-2 alpha) is typically associated with stress responses and causes a reduction in protein synthesis. However, we found high phosphorylated eIF-2 alpha (eIF-2 alpha[P]) levels in nonstressed pancreata of mice. Administration of glucose stimulated a rapid dephosphorylation of eIF-2 alpha. Among the four eIF-2 alpha kinases present in mammals, PERK is most highly expressed in the pancreas, suggesting that it may be responsible for the high eIF-2 alpha[P] levels found therein. We describe a Perk knockout mutation in mice. Pancreata of Perk(-/-) mice are morphologically and functionally normal at birth, but the islets of Langerhans progressively degenerate, resulting in loss of insulin-secreting beta cells and development of diabetes mellitus, followed later by loss of glucagon-secreting alpha cells. The exocrine pancreas exhibits a reduction in the synthesis of several major digestive enzymes and succumbs to massive apoptosis after the fourth postnatal week. Perk(-/-) mice also exhibit skeletal dysplasias at birth and postnatal growth retardation. Skeletal defects include deficient mineralization, osteoporosis, and abnormal compact bone development. The skeletal and pancreatic defects are associated with defects in the rough endoplasmic reticulum of the major secretory cells that comprise the skeletal system and pancreas. The skeletal, pancreatic, and growth defects are similar to those seen in human Wolcott-Rallison syndrome.

Reperfusion after global brain ischemia results initially in a widespread suppression of protein synthesis in neurons, which persists in vulnerable neurons, that is caused by the inhibition of translation initiation as a result of the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha). To identify kinases responsible for eIF2alpha phosphorylation [eIF2alpha(P)] during brain reperfusion, we induced ischemia by bilateral carotid artery occlusion followed by post-ischemic assessment of brain eIF2alpha(P) in mice with homozygous functional knockouts in the genes encoding the heme-regulated eIF2alpha kinase (HRI), or the amino acid-regulated eIF2alpha kinase (GCN2). A 10-fold increase in eIF2alpha(P) was observed in reperfused wild-type mice and in the HRI-/- or GCN2-/- mice. However, in all reperfused groups, the RNA-dependent protein kinase (PKR)-like endoplasmic reticulum eIF2alpha kinase (PERK) exhibited an isoform mobility shift on SDS-PAGE, consistent with the activation of the kinase. These data indicate that neither HRI nor GCN2 are required for the large increase in post-ischemic brain eIF2alpha(P), and in conjunction with our previous report that eIF2alpha(P) is produced in the brain of reperfused PKR-/- mice, provides evidence that PERK is the kinase responsible for eIF2alpha phosphorylation in the early post-ischemic brain.

The Drosophila melanogaster Gld gene has multiple and diverse developmental and physiological functions. We report herein that interactions among proximal promoter elements and a cluster of intronically located enhancers and silencers specify the complex regulation of Gld that underlies its diverse functions. Gld expression in nonreproductive tissues is largely determined by proximal promoter elements with the exception of the embryonic labium where Gld is activated by an enhancer within the first intron. A nuclear protein, GPAL, has been identified that binds the Gpal elements in the proximal promoter region. Regulation of Gld in the reproductive organs is particularly complex, involving interactions among the Gpal proximal promoter elements, a unique TATA box, three distinct enhancer types, and one or more silencer elements. The three somatic reproductive organ enhancers each activate expression in male and female pairs of reproductive organs. One of these pairs, the male ejaculatory duct and female oviduct, are known to be developmentally homologous. We report evidence that the other two pairs of organs are developmentally homologous as well. A comprehensive model to explain the full developmental regulation of Gld and its evolution is presented.

A family of protein kinases regulates translation in response to different cellular stresses by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha). In yeast, an eIF-2alpha kinase, GCN2, functions in translational control in response to amino acid starvation. It is thought that uncharged tRNA that accumulates during amino acid limitation binds to sequences in GCN2 homologous to histidyl-tRNA synthetase (HisRS) enzymes, leading to enhanced kinase catalytic activity. Given that starvation for amino acids also stimulates phosphorylation of eIF-2alpha in mammalian cells, we searched for and identified a GCN2 homologue in mice. We cloned three different cDNAs encoding mouse GCN2 isoforms, derived from a single gene, that vary in their amino-terminal sequences. Like their yeast counterpart, the mouse GCN2 isoforms contain HisRS-related sequences juxtaposed to the kinase catalytic domain. While GCN2 mRNA was found in all mouse tissues examined, the isoforms appear to be differentially expressed. Mouse GCN2 expressed in yeast was found to inhibit growth by hyperphosphorylation of eIF-2alpha, requiring both the kinase catalytic domain and the HisRS-related sequences. Additionally, lysates prepared from yeast expressing mGCN2 were found to phosphorylate recombinant eIF-2alpha substrate. Mouse GCN2 activity in both the in vivo and in vitro assays required the presence of serine-51, the known regulatory phosphorylation site in eIF-2alpha. Together, our studies identify a new mammalian eIF-2alpha kinase, GCN2, that can mediate translational control.

Genomic and cDNA clones homologous to the yeast GCN2 eIF-2alpha kinase (yGCN2) were isolated from Drosophila melanogaster. The identity of the Drosophila GCN2 (dGCN2) gene is supported by the unique combination of sequence encoding a protein kinase catalytic domain and a domain homologous to histidyl-tRNA synthetase and by the ability of dGCN2 to complement a deletion mutant of the yeast GCN2 gene. Complementation of Deltagcn2 in yeast by dGCN2 depends on the presence of the critical regulatory phosphorylation site (serine 51) of eIF-2alpha. dGCN2 is composed of 10 exons encoding a protein of 1589 amino acids. dGCN2 mRNA is expressed throughout Drosophila development and is particularly abundant at the earliest stages of embryogenesis. The dGCN2 gene was cytogenetically and physically mapped to the right arm of the third chromosome at 100C3 in STS Dm2514. The discovery of GCN2 in higher eukaryotes is somewhat unexpected given the marked differences between the amino acid biosynthetic pathways of yeast vs. Drosophila and other higher eukaryotes. Despite these differences, the presence of GCN2 in Drosophila suggests at least partial conservation from yeast to multicellular organisms of the mechanisms responding to amino acid deprivation.

The 5' untranslated regions (UTRs) of the Drosophila Ubx and Antp genes were tested for their ability to promote cap-independent translation initiation. The Ubx and the Antp 5' UTR were inserted between the CAT and lacZ coding sequences in a dicistronic gene and tested for IRES activity in transgenic Drosophila. Northern analysis of the mRNAs showed the presence of the predicted full-length dicistronic mRNAs. High CAT activity was expressed from the first cistron from all of the dicistronic constructs introduced into the fly genome. The dicistronic transgenic strains bearing the Ubx and Antp IRES elements expressed significant levels of beta-galactosidase (betaGAL) from the second cistron whereas little or no betaGAL was expressed in the controls lacking the IRESs. In situ analysis of betaGAL expression in the transgenic strains indicates that expression of the second cistron is spatially and temporally regulated. Although the developmental patterns of expression directed by the Antp and Ubx IRESs overlap, they exhibit several differences indicating that these IRESs are not functionally equivalent.

Phosphorylation of the translation initiation factor eIF-2alpha downregulates protein synthesis by sequestering the guanylate exchange factor eIF-2B. The importance of this regulation has been demonstrated in the context of stress and virally induced repression of protein synthesis but has not been investigated relative to the control of protein synthesis during development. Transgenic Drosophila strains bearing aspartic acid or alanine substitutions at the presumed regulatory phosphorylation site (Ser50) of Drosophila eIF-2alpha were established. The expression of the eIF-2alpha mutant transgenes, under the transcriptional control of the hsp70 promoter, was induced at various times during development to assess the developmental and biochemical effects. Flies bearing the aspartic acid eIF-2alpha mutant (HD) transgene displayed a slow growth phenotype and small body size. Repeated induction of the HD transgene resulted in cessation of development. In contrast, flies bearing the alanine eIF-2alpha mutant (HA) displayed a fast growth phenotype and females were significantly larger than nontransgenic control sisters. The HD transgenic flies exhibit a relatively lower level of global protein synthesis than the HA transgenic flies, although the difference is statistically insignificant.

The Glucose dehydrogenase (Gld) gene is highly expressed in the mature Drosophila reproductive tract. Unlike several other Drosophila genes which function in reproductive physiological processes, Gld is also expressed extensively in the developing reproductive tract during metamorphosis. Proximal promoter elements drive Gld expression in a variety of tissues throughout development, but not in the reproductive tract. Herein, we have identified a somatic reproductive organ enhancer complex (SREC) containing multiple redundant enhancer modules in Gld intron I (+639 to +3906 nt). The SREC, in combination with the Gld promoter, activates beta galactosidase reporter gene expression in both the developing and the mature reproductive tract. The SREC activates a heterologous hsp70 promoter in the ejaculatory duct, but not in other reproductive tract tissues, suggesting that the SREC acts synergistically with Gld promoter proximal elements. Through deletion analysis we have delimited a 361-nt region of the SREC that is involved in ejaculatory duct/oviduct-specific expression. The ejaculatory duct/oviduct enhancer retains the ability to activate expression in both the developing and the mature reproductive tract, suggesting that the same basic enhancer elements activate Gld expression during metamorphosis and in adults. A model of the evolution of Gld expression in the ejaculatory duct and oviduct is presented.

Heat shock of mammalian cells causes changes in initiation factor phosphorylation that likely contribute to or cause the translation reprogramming characteristic of heat shock. In these investigations we have carried out a parallel analysis of Drosophila, focusing on eIF-4E and eIF-2 alpha. eIF-4E plus associated proteins was purified from lysates by m7GTP-Sepharose chromatography. A minor fraction (< 10%) of eIF-4E is phosphorylated under normal growth conditions, and phosphorylation decreases during heat shock. Drosophila eIF-2 alpha has been identified by in vitro translation of T7 RNA polymerase-transcribed mRNA, and immunoblotting with anti-Drosophila eIF-2 alpha antiserum. 32P-labeling analysis (unfractionated cell lysates and immunoprecipitates) detects phosphorylated eIF-2 alpha, whose amount increases approximately 2-3-fold upon heat shock. Immunoblotting analysis of two-dimensional gel-resolved proteins to determine the mass fraction of eIF-2 alpha phosphorylated detects a single eIF-2 alpha spot in both normal temperature and heat shocked cells, indicating less than 5% phosphorylation after and before heat shock. Staining quantification is consistent with this low prevalence. A major phosphoprotein which copurifies with eIF-4E on m7GTP-Sepharose shows decreased overall phosphorylation and decreased association with eIF-4E following heat shock. Several distinctive characteristics of this phosphoprotein suggest it is Drosophila eIF-4B.

Drosophila melanogaster cDNA clones encoding the beta subunit of translation initiation factor 2 (eIF-2) were isolated and sequenced. The longest cDNA predicts a protein of 312 amino acids (aa), which possesses a putative RNA-binding motif and a highly charged N-terminal region composed of three basic polylysine blocks. The aa sequence comparison of D. melanogaster eIF-2 beta with its human and yeast counterparts demonstrates a high degree of similarity, especially within the C-terminal region. Northern analysis indicates quasi-constitutive expression of eIF-2 beta throughout D. melanogaster development.

A conserved palindromic sequence (Gpal) in the promoter region of the Drosophila Gld directs expression of a heterologous reporter gene in transgenic flies to the anterior spiracular glands of third instar larvae and to the ejaculatory bulb of adult males. The Gld gene is normally expressed at high levels in the anterior spiracular glands but is not expressed in the ejaculatory bulb of Drosophila melanogaster. However, Gld promoters from other Drosophila species contain the Gpal element and express glucose dehydrogenase (GLD) in the adult male ejaculatory bulb. A gene fusion composed of the D. melanogaster Gld promoter and the lacZ gene is expressed in the anterior spiracular glands of transgenic larvae. Mutations of the Gpal sequence element in this gene fusion block expression of beta-galactosidase in the anterior spiracular gland. Together these experiments demonstrate that Gpal is necessary and sufficient for tissue-specific expression in the anterior spiracular glands. Based upon the tissue distribution and function of GLD, it is speculated that expression of GLD in the anterior spiracular glands represents the ancestral state and that GLD expression in other tissues arose as a fortuitous consequence of a shared combinatorial regulatory network.

Genomic and cDNA clones encoding the Drosophila melanogaster alpha-subunit of translational initiation factor 2 (eIF-2 alpha) were isolated. The D. melanogaster eIF-2 alpha gene encodes a 341 amino-acid (aa) protein that shares 57 and 44% identity to its human and yeast homologues, respectively. The regulatory phosphorylation site at Ser50 is embedded in a segment of 19 conserved aa residues. Analysis of the genomic DNA and cDNA clones indicated that eIF-2 alpha is a single-copy gene and its coding region is interrupted by a 260-bp intron. The D. melanogaster eIF-2 alpha mRNA is 1350 nt in length and is expressed throughout development.

The tissue-specific expression patterns of glucose dehydrogenase (GLD) exhibit a high degree of interspecific variation in the adult reproductive tract among the species in the genus Drosophila. We chose to focus on the evolution of GLD expression and the evolution of the Gld promoter in seven closely related species in the melanogaster subgroup as a means of elucidating the relationship of changes in cis-acting regulatory elements in the Gld promoter region with changes in tissue-specific expression. Although little variation in tissue-specific patterns of GLD was found in nonreproductive tissues during development, a surprisingly high level of variation was observed in the expression of GLD in both developing and mature reproductive organs. In some cases this variation is correlated with changes in sequence elements in the Gld promoter which were previously shown to direct tissue-specific expression in the reproductive tract. In particular D. teissieri adult males do not express GLD in their ejaculatory ducts, atypical of the melanogaster subgroup species. The Gld promoter region of D. teissieri specifically lacks all three of the TTAGA regulatory elements present in D. melanogaster. The TTAGA elements were previously shown to direct reporter gene expression to the ejaculatory duct. Together these data suggest the absence or presence of the TTAGA elements may be responsible for variation in the absence or presence of GLD in the ejaculatory duct among species.

Putative cis-acting regulatory elements immediately upstream of the Gld promoter were identified by comparative analysis of three Drosophila species. A 509 bp region containing these elements and the Gld promoter region was shown to confer tissue-specific regulation to a reporter gene similar to the pattern observed for Gld mRNA and protein. A dispersed repeat with a core motif of TTAGA was also capable of directing the expression of a reporter gene to several epidermally derived tissues in which GLD is normally expressed. These tissues include male and female somatic reproductive organs. The TTAGA elements and a palindromic element act antagonistically to block expression of reporter gene in some tissues. Previously reported mutations of the heat shock response element resulted in the creation of three TTAGA elements. This mutated hsp70 promoter directs expression of a reporter gene to many of the same tissues as does the Gld TTAGA elements. We have found TTAGA elements near the promoter of two other genes which show an identical expression pattern in the male ejaculatory duct as Gld and the mutant hsp70.

During the preadult development of Drosophila melanogaster, the GLD (glucose dehydrogenase) gene (Gld) is expressed in a variety of tissues, including the immature reproductive tract. At the adult stage the expression of Gld becomes largely restricted to the reproductive tract of males and females. We examined the expression of GLD in the adult reproductive tract of 50 species in the genus Drosophila, as well as in those of a few representative species from four other closely related genera. GLD exhibits considerable organ-specific diversity in the reproductive tract of males and females. Among these species, five male GLD phenotypes and six female GLD phenotypes were found. In contrast, the preadult expression of GLD in representative species from each distinct adult pattern type was determined and found to be highly conserved in both the immature reproductive tract and non-reproductive organs. Moreover, the set of reproductive organs that express GLD during preadult development is equivalent to the sum of the five male and six female adult GLD phenotypes. To initially define the contribution of cis- versus trans-acting factors responsible for differences in adult GLD expression between two of these species--D. melanogaster and D. pseudoobscura--we transferred the D. pseudoobscura Gld to the genome of D. melanogaster and investigated its expression. GLD expression patterns of these transformants displayed characteristics that are unique to both species, suggesting the presence of both cis- and trans-acting differences between these two species.

Several lethal mutations were identified previously in the 84BD interval of the Drosophila melanogaster third chromosome (Lewis et al., 1980; Cavener et al., 1986b). We have examined the l(3)84Cd complementation group and found that mutants exhibit novel cuticular defects and die during larval development. The lethal phase occurs during the first larval molt or subsequently during the second instar larval stage; hence, we have named the gene stranded at second (sas). There are no apparent effects on the rate of development of embryos or first instar larvae. Second instar larvae which survive the molt exhibit a marked reduction in growth and eventually die as small second instar larvae. Incomplete penetrance in some weak sas alleles can yield fertile adults. In addition to the lethal phenotype, a segmentally repeated pattern of tanned spots is found within the ventral setal belts of mutant larvae. The position of the spots is always either between the fourth and fifth row of setae (cuticular projections) or between the first and second row of setae. The spots are adjacent to the muscle attachment sites in the setal belt region. Another common larval phenotype is the abnormal tanning of the ventral surface of the pharynx. The sas gene was cloned, and both the cuticular tanning and the larval lethal phenotypes were complemented by P-element-mediated transformation with a genomic DNA-cDNA construct. Three major sas transcripts are expressed throughout development in cuticle secreting epidermal tissues. The sas transcripts show stage- and tissue-specific patterns of expression with switches in transcript patterns occurring at the molts. The inferred 1348-amino-acid sequence suggests that sas encodes a cell surface protein which functions as a receptor. The putative extracellular region contains four tandem repeats of a cysteine-rich motif which is similar to a cysteine pattern present in procollagen and in thrombospondin. Following this region are at least three copies of a fibronectin type III class repeat. The short (35 amino acids) intracellular domain contains a sequence (NPXY) that has been implicated in endocytosis via coated pits.

The ability to transfer genes from one species to another provides a powerful method to study genetic regulatory differences between species in a homogeneous genetic background. A survey of several transgenic animal experiments indicates that the vast majority of regulatory differences observed between species are due to differences in the cis-acting elements associated with the genes under study. A corollary is that in almost all cases the host species provides the necessary regulatory proteins for expression of the transgenes in specific tissues in which the endogenous homolog is not expressed. Although the details of the cis-acting differences are unknown for most cases, it appears that these differences may consist of the acquisition or loss of unique elements or subtle variation of conserved elements. It is unknown whether much of this variation is directly related to adaptive evolution. The identification of the promoter/enhancer elements responsible for these differences is an important first step in examining the functional significance of this variation.

Sequence comparison of Drosophila melanogaster glucose dehydrogenase, Escherichia coli choline dehydrogenase, Aspergillus niger glucose oxidase and Hansenula polymorpha methanol oxidase indicates that these four diverse flavoproteins are homologous, defining a new family of proteins named the GMC oxidoreductases. These enzymes contain a canonical ADP-binding beta alpha beta-fold close to their amino termini as found in other flavoenzymes. This domain is encoded by a single exon of the D. melanogaster glucose dehydrogenase gene.

The presence of estrogen receptor (ER) is a well-known predictor of clinical outcome in human breast cancer. We examined the ER gene in 26 primary breast cancers (14 ER-positive, 12 ER-negative) to determine if alterations of the gene are associated with the ER-negative status. In tumor biopsies and peripheral blood DNA obtained from the same patients we analyzed the ER exon structure using polymerase chain reaction amplification, restriction endonuclease digestion, and agarose gel electrophoresis. All blood and tumor samples, regardless of ER status, showed a complete set of eight exons of normal sizes, ruling out deletions or rearrangements of the ER gene in excess of +/- 20 nucleotides. Previous reports indicate that the two-allele ER PvuII polymorphism could be associated with ER expression in breast cancer (Hill et al., Cancer Res., 49: 145-148, 1989) as well as with patient age at time of tumor diagnosis (Parl et al., Breast Cancer Res. Treat., 14: 57-64, 1989). We localized the PvuII polymorphism in intron 1, 0.4 kilobase upstream of exon 2. Sequence analysis showed the polymorphism to result from a point mutation (T----C) at the fifth position of the restriction site (CATCTG). We determined the PvuII restriction fragment-length polymorphism genotype in 257 primary breast cancers and 140 peripheral blood DNA samples obtained from women without breast cancer. The results indicate that the PvuII polymorphism is not associated with ER content or patient age at tumor diagnosis.

The sex-determination genes of Drosophila act to repress the developmental pathway for the internal somatic reproductive organs of the opposite sex. By misregulating this pathway during preadult development, the organ-specific expression pattern of the glucose dehydrogenase gene (Gld) in the reproductive tract of adult flies has been changed without a concomitant sexual transformation of the reproductive organs. Misregulation of the tra, tra-2, and dsx genes leads to very similar patterns of ectopic expression of Gld. The induced ectopic patterns of Gld expression at the adult stage occur in a small subset of organs which all normally express the Gld gene during their morphogenesis. These ectopic patterns are irrevocably set during late larval-early pupal development. The normal pattern of Gld expression in several other Drosophila species is quite similar to the ectopic patterns which we have generated in D. melanogaster, suggesting that the interspecific variation in Gld expression may result from variation in the expression of the sex-determination genes.

The estrogen receptor (ER) is a nuclear protein with a hormone- and a DNA-binding domain. We examined the DNA binding of ER in MCF-7 cells and 79 primary breast cancers by gel mobility shift assay using as a probe the estrogen response element (ERE). The mobility shift assay showed saturable, specific binding of ER to ERE in crude, high molar extracts containing greater than or equal to 4 mg/ml protein. Nonspecific binding was reduced by increasing concentrations of poly(deoxyinosidylate.deoxycytidylate) and shortening of the ERE probe from 35 to 15 base pairs. In the presence of Mg2+ the ER-ERE complex formation was hormone dependent at 22 degrees C but not at 37 degrees C. In the absence of Mg2+ estradiol was not necessary for ER-ERE complex formation. Correlation of the mobility shift assay with the hormone-binding (E2) assay showed agreement in 55 of the 79 tumors. Both assays were positive (E2 +/ERE+) in 35 cases and both were negative (E2-/ERE-) in 20 cases. In 11 tumors the hormone-binding assay was positive and the mobility shift assay negative (E2+/ERE-), suggesting an alteration of the DNA-binding domain. In 13 cancers the hormone-binding assay was negative and the mobility shift assay positive (E2-/ERE+) suggesting an alteration of the hormone-binding domain. By performing both hormone- and DNA-binding assays of ER and the hormone-binding assay of progesterone receptor (PR), we found the following subgroups of breast cancer: (a) E2+/ERE+/PR+, (b) E2+/ERE+/PR-, (c) E2+/ERE-/PR+, (d) E2+/ERE-/PR-, (e) E2-/ERE+/PR+, (f) E2-/ERE+/PR-, (g) E2-/ERE-/PR-. The simultaneous determination of 17 beta-estradiol and ERE binding may provide a better definition of the ER status of individual tumors and prove useful in refining endocrine therapy of patients with breast cancer.

Sequences flanking translational initiation and termination sites have been compiled and statistically analyzed for various eukaryotic taxonomic groups. A few key similarities between taxonomic groups support conserved mechanisms of initiation and termination. However, a high degree of sequence variation at these sites within and between various eukaryotic groups suggest that translation may be modulated for many mRNAs. Multipositional analysis of di-, tri-, and quadrinucleotide sequences flanking start/stop sites indicate significant biases. In particular, strong tri-nucleotide biases are observed at the -3, -2, and -1 positions upstream of the start codon. These biases and the interspecific variation in nucleotide preferences at these three positions have lead us to propose a revised model of the interaction of the 18S ribosomal RNA with the mRNA at the site of translation initiation. Unusually strong biases against the CG dinucleotide immediately downstream of termination codons suggest that they may lead to faulty termination and/or failure of the ribosome to disassociate from the mRNA.

The importance to in vivo translation of sequences immediately upstream of the Drosophila alcohol dehydrogenase (Adh) start codon was examined at two developmental stages. Mutations were introduced into the Adh gene in vitro, and the mutant gene was inserted into the genome via germ line transformation. An A-to-T substitution at the -3 position did not affect relative translation rates of the ADH protein at the second-instar larval stage but resulted in a 2.4-fold drop in translation of ADH at the adult stage. A second mutant gene, containing five mutations in the region -1 to -9, was designed to completely block translation initiation. However, transformant lines bearing these mutations still exhibit detectable ADH, albeit at substantially reduced levels. The average fold reduction at the second-instar larval stage was 5.9, while at the adult stage a 12.5-fold reduction was observed.

The Gld gene of Drosophila melanogaster is transiently expressed during every stage of development. The temporal pattern of Gld expression is highly correlated with that of ecdysteroids. Exogeneous treatment of third instar larvae with 20-hydroxyecdysone induces the accumulation of Gld mRNA in the hypoderm and anterior spiracular gland cells. During metamorphosis Gld is expressed in a variety of tissues derived from the ectoderm. In the developing reproductive tract, Gld mRNA accumulates in the female spermathecae and oviduct and in the male ejaculatory duct and ejaculatory bulb. These four organs are derived from closely related cell lineages in the genital imaginal disc. Since the expression of Gld is not required for the development of these reproductive structures, this spatial pattern of expression is most likely a fortuitous consequence of a shared regulatory factor in this cell lineage. At the adult stage a high level of the Gld mRNA is only observed in the male ejaculatory duct.

The glucose dehydrogenase genes (Gld) of Drosophila melanogaster, of D. pseudoobscura, and of D. virilis have been isolated and compared with each other in order to identify conserved and divergent aspects of their structure and expression. The exon/intron structure of Gld is conserved. The Gld mRNAs are similar, with a range of 2.6-2.8 kb among the three species. All three species exhibit peaks of Gld expression during every major developmental stage, although considerable variation in the precise timing of these peaks exists between species. Interspecific gene transfer experiments demonstrate that the regulation and function of the D. pseudoobscura Gld is similar enough to the homologous gene in D. melanogaster to substitute for its essential role in the eclosion process. Comparison of the putative promoter sequences has identified both shared and divergent sequence elements which are likely responsible, respectively, for the conserved and divergent patterns of expression observed. The entire coding sequences of the pseudoobscura and melanogaster Gld genes are presented and shown to encode a 612-amino-acid pre-protein. The inferred amino acid sequences are 92% conserved between the two species. In general the intronic regions of Gld are unusually well conserved.

A survey of 29 laboratory populations of Drosophila melanogaster for glutamate oxaloacetate transaminase (GOT) variation revealed that the Got-1 locus was polymorphic in three stocks recently collected from nature. The Got-1 locus was fixed for the same allozyme in the remaining 26 laboratory populations. Testcross matings to multiply marked stocks established that Got-1 is located at 4.8 centimorgans on chromosome 2.

The temporal patterns of glucose dehydrogenase (Gld) and alcohol dehydrogenase (Adh) expression in Drosophila are correlated positively and negatively, respectively, with ecdysterone titers during the late third instar. In mutant l(3)ecdysone-1ts (ecd-1) larvae, the normal peak of Gld mRNA late in the third instar is not expressed. Conversely, the normal decrease in Adh mRNA at this stage fails to occur in ecd-1. These two abnormal patterns can be reversed by treatment with exogenous ecdysterone. Premature exposure of wild type mid-third instar larvae to ecdysterone also results in the rapid accumulation of Gld mRNA and signals the repression of Adh mRNA. The observed decrease in Adh mRNA expression is accompanied by a transient switch in promoter usage from proximal to distal transcription start sites, which normally occurs later in the third instar.

Two strains of Drosophila melanogaster derived from different geographical localities were subjected to selection for fast and slow developmental rate. Four enzyme polymorphisms were monitored for their response to such selection. Significant changes in allele frequencies were effected for the alphaGpdh and 6Pgd polymorphisms under fast developmental rate selection. The strain from Rhode Island exhibited a correlated change in body weight during developmental rate selection, whereas the Georgia strain did not.

Seasonal patterns of allozyme variation are examined for 12 polymorphic enzyme loci in Drosophila melanogaster. The data derive from a total of 56 samples taken from a natural population in the Summer and Fall of 1978 and 1979. Samples were obtained at approximately five-day intervals and assayed for 6-phosphogluconate dehydrogenase (6Pgd), phosphoglucomutase (Pgm) and glucose-6-phosphate dehydrogenase (G6pd). The remaining nine enzymes were assayed in an average of eight samples per season. None of the loci exhibit regular seasonal cycles of gene-frequency change, although 6Pgd does show significant, but irregular, frequency oscillations. There is also little evidence for gene-frequency differences between years, although 6Pgd is again exceptional in showing significant frequency changes between years. In addition, genotypic frequency distributions are usually consistent with random mating expectations. With the notable exception of 6Pgd, the data give a strong impression of gene-frequency homogeneity within and among years, despite obvious seasonal changes in climate and in the distribution of breeding sites.

Glucose dehydrogenase (GO) was found to be expressed during the pupal stage in both sexes of Drosophila melanogaster, but is limited to the male ejaculatory duct at the adult stage. During copulation GO is transferred from males to females. Mutational analysis of the Go locus indicates that a single structural gene encodes the pupal and ejaculatory duct GO. Thus an example of an enzyme structural gene switching from non-sex-limited to sex-limited expression has been found. Go mutants are recessive lethals exhibiting a late pupal effective lethal phase. These mutants can be rescued by excising the anterior end of the pupal case 0-2 days prior to the normal adult emergence time. It appears that the function of GO in pupae is to aid in the degradation of the puparium cuticle in preparation for the eclosion of the adult.

The in vivo flux of carbon through the pentose shunt is investigated as a function of different 6-phosphogluconate dehydrogenase (6Pgd) and glucose-6-phosphate dehydrogenase (G6pd) genotypes by using differential radioactive labeling of the C-1 and C-6 positions of glucose. Alternative 6Pgd-G6pd genotypes are shown to differ in relative in vivo carbon flux through the pentose shunt. The relative in vitro specific activity differences between the 6PgdSS and 6PgdFF genotypes appear to be primarily responsible for these differences. In addition, the pentose-shunt activity is correlated with the rate of lipid synthesis. This correlation is consistent with the major metabolic function of the pathway, which is to produce NADPH for lipid synthesis. Taken together, the results of these experiments show that different genotypes of 6Pgd are associated with measurable biochemical and physiological differences. Higher order phenotypic differences of this kind must be demonstrated to support the hypothesis that natural selection can discriminate among allozymes of a given genetic locus.

A glucose oxidase (GO) has been identified in the ejaculatory duct of male Drosophila melanogaster. Evidence is given that this enzyme was previously misidentified as HEX-1. Genetic analysis indicates that the Go structural gene is located on the third chromosome at 48 + or - 0.5 cm. Go is polymorphic in males in populations of D. melanogaster and D. simulans located in Athens, Georgia. Two other hexose enzymes have also been tentatively identified for the first time in Drosophila. These are NAD(P)-glucose dehydrogenase (GODH) and NAD-gluconate dehydrogenase (GNDH). GODH and GNDH are found in both males and females and may circumvent the initial steps in the pentose shunt.

The glucose dehydrogenase gene (Gld) in Drosophila melanogaster exhibits a unique spatial and temporal pattern of expression. GLD expression switches from a non-sex-limited state at the pupal stage to a male-limited state at the adult stage. At the adult stage, the enzyme is restricted to the ejaculatory duct. Within the genus Drosophila, the ejaculatory duct has undergone a simple morphological divergence. In order to determine whether correlated changes in GLD expression had occurred, GLD activity during the pupal and adult stages was determined for several Drosophila species. It was found that virtually all of the species exhibit pupal GLD activity, whereas only those species with an expanded ejaculatory duct express male-limited GLD. The results of interspecific genital imaginal disc transplantation experiments indicate that the expanded morphology and GLD expression do not require any species- or sex-specific diffusible factors. An apparent regulatory polymorphism exists within the D. takahashii species with respect to male-limited GLD expression.

A human estrogen receptor (ER) cDNA probe was used to examine genomic DNA extracted from 59 primary invasive breast cancers. The tumors were also studied histopathologically, and their ER status was assessed by hormone-binding assay and immunohistochemical analysis. Southern blots of genomic DNA samples digested with restriction endonucleases (BamHI, EcoRI, HindIII, PvuII, XbaI) revealed identical restriction fragments for each tumor, indicating preservation of gross ER gene integrity regardless of ER status, clinical stage, or histopathologic appearance. Digestion with PvuII identified a single, two-allele polymorphism with band(s) at 1.6 and/or 0.7 kb. The homozygous 1.6 kb pattern was present in 14 (24%) patients, the heterozygous 1.6/0.7 kb pattern in 29 (49%) patients and the homozygous 0.7 kb pattern in 16 (27%) patients. The PvuII restriction fragment length polymorphism (RFLP) within the ER gene showed no correlation with the results of the ER binding assay, the immunohistochemical analysis, clinical stage, or histopathologic appearance. A significant correlation was found between the genotypes and patient age at the time of tumor diagnosis. Tumors with the homozygous 1.6 kb and the heterozygous 1.6/0.7 kb patterns were observed in older women with mean ages of 64.6 and 64.4 years, respectively. In contrast, patients with tumors containing the homozygous 0.7 kb pattern were significantly younger, with a mean age of 50.4 years (p-value = 0.0024). The mechanism by which the homozygous 0.7 kb genotype is associated with breast cancer in the premenopausal age group is unknown.

Four replicate populations of Drosophila melanogaster, two reared on medium supplemented with ethanol and two reared on standard medium, were electrophoretically monitored for 28 generations. During the first 12 generations, allelic, genotypic and gametic frequencies were determined for eight polymorphic enzymes: GOT, alpha-GPDH, MDH, ADH, TO, E6, Ec and ODH. Samples from generation 18 and 28 were electrophoretically typed for ADH and alpha-GPDH. In addition, samples from generation 27 were analyzed for the presence of inversion heterozygotes. The experimental results showed rapid gene-frequency divergence between control and treatment populations at the Adh locus in a direction consistent with the activity hierarchy of Adh genotypes. Gene-frequency divergence between control and treatment populations also occurred at the alpha-Gpdh locus, although the agreement among replicates appeared to have broken down by generation 28. No differential gene-frequency change occurred at any of the six remaining marker loci. Furthermore, values of linkage disequilibria among all linked pairs of genes were initially small and remained small throughout the course of the experiment. Taking these facts into account, it is argued that the gene-frequency response observed at ADH is most probably caused by selection at the Adh locus. The gene frequency response at alpha-Gpdh can also be be accounted for in terms of the effect of ethanol on energy metabolism, although other explanations cannot be excluded.

We transcribed a cDNA clone of the human estrogen receptor (ER) with T7 RNA polymerase. The 32P-cRNA transcript complementary to ER mRNA was hybridized to poly(A)+ RNA from human uterus and revealed a single band of approximately 4.2 kilobases. No hybridization was seen with the cRNA probe of the opposite orientation. Hybridization of total RNA from calf and rat uterus yielded a single band at approximately 3.8 kilobases for both species. Total RNA from rat spleen did not hybridize. A 35S-labeled cRNA probe was prepared for in situ hybridization of ER mRNA in human uterus and spleen. Autoradiographic signal was present over endometrial epithelium, stromal cells of the lamina propria, and smooth muscle cells of the myometrium but was absent from sections of spleen. The ER mRNA hybridization label was located over cytoplasm and nuclei of uterine target cells.

A reanalysis of the 84B3 to 84D3,5 region of the polytene chromosomes of Drosophila melanogaster has led to the identification and localization of 16 genes. These genes include 11 vital loci, four genes exhibiting nonlethal visible mutant phenotypes and one gene encoding a nonessential enzyme. The identity of the gene products of two of the vital genes has been determined to be alpha-tubulin and glucose dehydrogenase (Gld). Three newly identified genes, sticking (stk), half out (hat) and trapped (ted), as well as Gld are required for eclosion. Among the nonessential genes are roughened eye (roe) and ruffed eye (rue), which affect eye texture. The roe phenotype is greatly enhanced by deletions that simultaneously remove roe and an unidentified locus in 84E. Mutations in another nonessential gene, rotund (rn), are characterized by pattern deletions of most adult appendages.

The previously presented consensus sequence for eukaryotic translation initiation sites by Kozak was derived substantially from vertebrate mRNA sequences. Drosophila nuclear genes exhibit a significantly different translation start consensus sequence. These differences probably do not represent mechanistic differences in translation initiation inasmuch as both taxa exhibit identical preferences and restrictions at the crucial -3 position. Using more conservative criteria for the assignment of consensus the following consensus sequences were derived: vertebrate--CANCAUG and Drosophila--CAAAACAUG.