Translation initiation in Drosophila melanogaster is reduced by mutations upstream of the AUG initiator codon.

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.

Ultrabithorax and Antennapedia 5' untranslated regions promote developmentally regulated internal translation initiation.

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.

Binding analysis of the estrogen receptor to its specific DNA target site in human breast cancer.

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.

Analysis of the PvuII restriction fragment-length polymorphism and exon structure of the estrogen receptor gene in breast cancer and peripheral blood.

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.

GMC oxidoreductases. A newly defined family of homologous proteins with diverse catalytic activities.

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 Response of Enzyme Polymorphisms to Developmental Rate Selection in DROSOPHILA MELANOGASTER.

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.

Dynamics of correlated genetic systems. IV. Multilocus effects of ethanol stress environments.

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.

Temporal Stability of Allozyme Frequencies in a Natural Population of DROSOPHILA MELANOGASTER.

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.

A rehabilitation of the genetic map of the 84B-D region in Drosophila melanogaster.

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.

Complex organization of promoter and enhancer elements regulate the tissue- and developmental stage-specific expression of the Drosophila melanogaster Gld gene.

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.

Developmental expression of the glucose dehydrogenase gene in Drosophila melanogaster.

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.

A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2alpha.

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.

Isolation of the gene encoding the Drosophila melanogaster homolog of the Saccharomyces cerevisiae GCN2 eIF-2alpha kinase.

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.

Tissue-specific regulatory elements of the Drosophila Gld gene.

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.

Isolation and characterization of the Drosophila melanogaster gene encoding translation-initiation factor eIF-2 beta.

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.

Isolation and characterization of the Drosophila melanogaster eIF-2 alpha gene encoding the alpha subunit of translation initiation factor eIF-2.

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.

Detection of estrogen receptor mRNA in human uterus.

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.

Mutations at the Ser50 residue of translation factor eIF-2alpha dominantly affect developmental rate, body weight, and viability of Drosophila melanogaster.

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.