One protein, two enzymes.

Two enzymes, designated, E-2 and E-2', catalyze different oxidation reactions of an aci-reductone intermediate in the methionine salvage pathway. E-2 and E-2', overproduced in Escherichia coli from the same gene, have the same protein component. E-2 and E-2' are separable on an anion exchange column or a hydrophobic column. Their distinct catalytic and chromatographic properties result from binding different metals. The apo-enzyme, obtained after metal is removed from either enzyme, is catalytically inactive. Addition of Ni2+ or Co2+ to the apo-protein yields E-2 activity. E-2' activity is obtained when Fe2+ is added. Production in intact E. coli of E-2 and E-2' depends on the availability of the corresponding metals. These observations suggest that the metal component dictates reaction specificity.

Linkage between oligomerization and DNA binding in Drosophila doublesex proteins.

The doublesex gene of Drosophila melanogaster encodes DSXM protein in males and DSXF protein in females. Dimers of each protein bind a DNA site from which DSXM represses and DSXF activates transcription. Amino acids 1-397 are identical between the proteins and include a domain (DBD) for both DNA binding and protein oligomerization. The remaining nonhomologous and therefore sex-specific C-termini include an essential part of a second oligomerization domain. We have used mobility shift assays to investigate the effects these three oligomerization domains (DBD and two sex-specific) have on DSX dimerization and DNA binding. The intrinsic DNA binding affinities of DSXM and DSXF dimers are indistinguishable from each other (0.17 +/- 0.04 nM) and slightly lower than that of DBD dimers (0.48 nM). In contrast, the dimerization dissociation constants of DSXM (0.05 +/- 0.02 nM) and DSXF (0.16 +/- 0.05 nM) are slightly different, but 4 orders of magnitude lower than that of DBD (430 nM). Thus sequences outside of DBD, presumably the sex-specific oligomerization domains, have substantial effects on apparent DNA binding affinity through thermodynamically linked effects on dimerization of full-length proteins. Further, when two DNA binding sites are adjacent, DBD dimers show no binding cooperativity, whereas full-length dimers bind with 2-fold different cooperativity (DSXF, k12 = 2.6; DSXM, k12 = 5.4). This suggests that the sex-specific domains may have a second effect on DNA binding, namely, an effect on binding cooperativity that depends on the number and arrangement of DNA sites.

DNA binding by the male and female doublesex proteins of Drosophila melanogaster.

Drosophila yolk protein genes are regulated by doublesex male protein (DSXM) in males and doublesex female protein (DSXF) in females. Both proteins bind to the same DNA sites from which DSXM represses and DSXF activates transcription. The proteins are identical through 397 NH2-terminal amino acids that include domains for oligomerization and DNA binding. The remaining COOH termini are sex-specific and include an essential part of a second oligomerization domain. We report here mobility shift assays that examine the DNA binding properties of purified DSXM and DSXF. Dimers of DSXM and DSXF bind to a regulatory site, dsxA, with the same affinity (Kapp = 0.2 nM), specificity (specific/nonspecific approximately 1.2 x 10(4)), and dependence on monovalent and divalent cations. The DNA association rate constants also are indistinguishable (kon = 4.6 x 10(6) M-1 s-1) as are the several terms of the dissociation reaction. Dissociation has an intrinsic rate of koff = 5.1 x 10(-4) s-1 and other rate terms that depend on the free concentration of specific DNA binding sites (2.4 x 10(4) M-1 s-1) or nonspecific binding sites (2.4 M-1 s-1). This first order dependence on unbound DNA suggests that a direct transfer between DNAs is likely to occur when DSX proteins search for specific sites in the many short open DNA regions of chromatin. Overall, dimer binding to individual DNA sites appears to be determined by the sex-nonspecific part of the two proteins. We infer that the sex-specific oligomerization domains play roles in binding cooperativity to multiple DNA sites or in other protein:protein interactions.

DNA binding specificities of YPF1, a Drosophila homolog to the DNA binding subunit of human DNA-dependent protein kinase, Ku.

YPF1, a heterodimeric protein from Drosophila melanogaster, is a homolog to Ku, the DNA binding subunit of human DNA-dependent protein kinase. This kinase is crucial in transcriptional activation, V(D)J recombination, double-strand break repair, and both topoisomerase and helicase activities. To investigate functional homology between YPF1 and Ku, we examined DNA binding properties of YPF1. Like Ku, at 100 mM KCl, YPF1 binding has no detectable DNA sequence specificity, requires a DNA terminus, and has a concentration-dependent stoichiometry consistent with subsequent translocation along DNA. YPF1 differs from Ku by having a 10(5)-fold higher affinity. At 400 mM KCl, YPF1 still prefers DNA termini but shows binding specificities not observed previously with Ku. In descending order of affinity, YPF1 binds to: specific DNA sequences with a specific polarity and spacing relative to DNA termini; nonspecific linear DNA; and circular DNA. At this higher ionic strength, binding stoichiometry is concentration independent, indicating that YPF1 remains bound to ends. These results demonstrate a strong functional homology between YPF1 and Ku at physiological ionic strength. The strong binding of YPF1 has also allowed us to detect underlying binding specificities that may be specific to YPF1 and its function.

Sex-specific and non-sex-specific oligomerization domains in both of the doublesex transcription factors from Drosophila melanogaster.

The doublesex gene of Drosophila melanogaster encodes the alternatively spliced, sex-specific transcription factors DSXM and DSXF. These factors regulate male- and female-specific transcription of many genes. For example, female-specific transcription of the yolk protein 1 gene is regulated by DSXM repression in males and DSXF activation in females. In this study we used in vitro interaction assays and the in vivo yeast two-hybrid method to identify and examine oligomerization domains of the DSX proteins. A 66-amino-acid segment common to both proteins (amino acids 39 to 104) contains a sequence-specific DNA binding domain and an oligomerization domain (OD1). The OD1 domain oligomerizes up to at least a pentamer, but only dimers bound to a palindromic regulatory site in the yolk protein 1 gene are detected. Both subunits of the OD1 dimer are in contact with DNA. Another segment of each protein (amino acids 350 to 412 for DSXF and 350 to 427 for DSXM) contains a second oligomerization domain (OD2F and OD2M, respectively). The OD2 domains have both sex-specific and non-sex-specific sequences which are necessary for oligomerization. On the basis of sequence analysis, we predict that OD2 oligomerizes through coiled-coil interactions. We speculate that the common function of OD1 and OD2 is to oligomerize the full-length proteins, whereas their specialized functions are to form a dimeric DNA binding unit and a sex-specific transcriptional activation or repression unit.

Purification and physical properties of the male and female double sex proteins of Drosophila.

The double sex gene (dsx) encodes two proteins, DSX(M) and DSX(F), that regulate sex-specific transcription in Drosophila. These proteins bind target sites in DNA from which the male-specific DSX(M) represses and the female-specific DSX(F) activates transcription of yolk protein (Yp) genes. We investigated the physical properties of these DSX proteins, which are identical in their amino-terminal 397 residues but are entirely different in their carboxyl-terminal sequences (DSX(F), 30 amino acids; DSX(M), 152 amino acids). DSX(M) and DSX(F) were overexpressed in cultured insect cells and purified to near homogeneity. Gel filtration chromatography and glycerol gradient sedimentation showed that at low concentrations both proteins are dimers of highly asymmetrical shape. The axial ratios are approximately 18:1 (DSX(M), 860 X 48 angstroms; DSX(F), 735 X 43 angstroms). At higher concentrations, the proteins form tetramers. Through use of a novel, double crosslinking assay (protein-DNA plus protein-protein), we demonstrated that a DNA regulatory site binds to both monomers of the DSX dimer and to only two monomers of the tetramer. Furthermore, binding another DNA molecule to what we presume is the second and identical site in the tetramer dramatically shifts the equilibrium from tetramers to dimers. These oligomerization and DNA binding properties are indistinguishable between the male and female proteins.

Regulation of Drosophila yolk protein genes by an ovary-specific GATA factor.

The divergently transcribed yolk protein genes (Yp1 and Yp2) of Drosophila melanogaster are expressed only in adult females, in fat body tissue and in ovarian follicle cells. Using an in vitro transcription assay, we have identified a single 12-bp DNA element that activates transcription from the promoters of both Yp genes. In vivo, this regulatory element is tissue specific: it activates transcription of Yp1 and Yp2 reporter genes in follicle cells but has no detectable effect in fat body or other tissues. The sequence of the element consists of two recognition sites for the GATA family of transcription factors. We show that among the Drosophila genes known to encode GATA factors, only dGATAb is expressed in ovaries. The single transcript that we detect in ovaries is alternatively spliced or initiated to produce an ovary-specific isoform of the protein. Bacterially expressed dGATAb binds to the 12-bp element; a similar binding activity is also present in the Kc0 nuclear extracts used for in vitro transcription assays. These in vitro and in vivo results lead us to propose that dGATAb makes several developmentally regulated products, one of which is a follicle cell-specific protein activating transcription of Yp1 and Yp2 from a known regulatory element.

Three protein binding sites form an enhancer that regulates sex- and fat body-specific transcription of Drosophila yolk protein genes.

Transcription of the Drosophila yolk protein (Yp) genes is regulated by the somatic sex determination pathway. A gene at the bottom of this pathway, doublesex, encodes the female-specific DSXF and male-specific DSXM proteins that bind to and regulate transcription from several sites in the Yp genes. We report site-directed mutagenesis, protein binding and germline transformation experiments that identify and characterize the activity of a single binding site (dsxA) for the doublesex proteins and two binding sites for other regulatory proteins. A single copy of the three sites is sufficient to direct the sex and fat body specificities of Yp transcription. The sites form an enhancer with two strongly synergistic enhancer elements. One element (22 bp) consists of dsxA and an overlapping site, bzip1, that binds the DmC/EBP (slbo) protein, a member of the bZIP family of transcriptional activators. The other element is an 11 bp binding site (ref1) for an unknown protein. Tissue-specific activation requires strong cooperation between the ref1 site and the bzip1 or dsxA sites. Sex specificity is regulated exclusively by the dsxA site which connects the sex determination pathway to the target gene through DSXM repression and DSXF activation.

Integrating sex- and tissue-specific regulation within a single Drosophila enhancer.

We have investigated the integration of sex- and tissue-specific transcriptional regulation in Drosophila. A single copy of the o-r enhancer from yolk protein genes directs female- and fat body-specific transcription. It consists of four protein-binding sites: dsxA, which binds male (DSXM) and female (DSXF) proteins encoded by the doublesex gene; aef1, which binds the AEF1 repressor; bzip1, which binds the DmC/EBP activator encoded by the slbo gene; and ref1, which binds an unknown activator. Multimeric and mutated binding sites were used in protein binding, germ-line transformation, and genetic experiments to examine the independent and combinatorial activities of the proteins and DNA sites. DSXF activates from dsxA by sterically excluding AEF1 repressor from the aef1 site and synergistically activating transcription together with a protein at bzip1. Sex specificity in fat bodies arises from the opposite effect of DSXM, which represses activity of the protein at bzip1. Tissue specificity is regulated by all four DNA sites. Separately, bzip1 and ref1 activate transcription in ovarian somatic cells and all nongonadal tissues, respectively, whereas together they activate only in fat bodies. The aef1 site represses ectopic transcription in ovaries and dsxA antirepresses this activity in fat bodies. Thus, in the organism, ref1 and bzip1 act combinatorially to direct the fundamental tissue specificity, aef1 and dsxA modulate this tissue specificity, and dsxA adds sex specificity.

GAGA factor and TBF1 bind DNA elements that direct ubiquitous transcription of the Drosophila alpha 1-tubulin gene.

Three DNA regions (TE1, TE2 and the intron) regulate the ubiquitous expression of the alpha 1-tubulin gene of Drosophila melanogaster. In this report, we identify two proteins that bind these DNA regions. One is the previously characterized GAGA transcription factor and the other is a newly identified 62 kDa polypeptide, TBF1 (TE1-binding factor 1). Purified GAGA factor binds three sites in TE2 and at least three in the intron. TBF1 was purified from embryos and binds to both TE1 and TE2. Together, the two proteins produce the same DNase I footprints in TE1 and TE2 as does a nuclear extract that transcribes the gene accurately. These footprints cover most of the TE1 and TE2 DNA. Moreover, one binding site for each protein coincides with a site that activates transcription in vitro. The characteristics of the GAGA factor and the genes it regulates suggest roles these two proteins are likely to play in regulating ubiquitous expression.

Insulating DNA directs ubiquitous transcription of the Drosophila melanogaster alpha 1-tubulin gene.

We identify DNA regions that are necessary for the ubiquitous expression of the Drosophila melanogaster alpha 1-tubulin (alpha 1t) gene. In vitro transcription showed that two upstream regions, tubulin element 1 (TE1 [29 bp]) and tubulin element 2 (TE2 [68 bp]), and a downstream region activate transcription. Germ line transformation demonstrated that these three regions are sufficient to direct the alpha 1t core promoter to begin transcribing at the stage of cellular blastoderm formation and to continue thereafter at high levels in all tissues and developmental stages. Remarkably, mutation of any one of these regions results in high sensitivity to chromosomal position effects, producing different but reproducible tissue-specific patterns of expression in each transformed line. None of these regions behaves as an enhancer in a conventional germ line transformation test. These observations show that these three regions, two of which bind the GAGA transcription factor, act ubiquitously to insulate from position effects and to activate transcription. The results also provide vectors for ubiquitous expression of gene products and for examining silencer activities.

Yolk protein factor 1 is a Drosophila homolog of Ku, the DNA-binding subunit of a DNA-dependent protein kinase from humans.

Yolk protein factor 1 (YPF1) is a heterodimeric DNA-binding protein from Drosophila melanogaster. In this report, we describe evidence that YPF1 is a homolog of Ku, a human autoimmune antigen that is the DNA-binding subunit of a DNA-dependent protein kinase. In vitro this kinase phosphorylates several transcription factors and, at the time of transcription initiation, the carboxyl-terminal domain of RNA polymerase II. We find that a cDNA clone for the smaller subunit (beta) of YPF1 encodes a 72-kDa protein that has extensive homology to the smaller subunit of the heterodimeric Ku protein (24% identity, 51% similarity over the entire 631 amino acid length). Further, the larger YPF1 subunit (alpha) shares immunological epitopes with the larger subunit of Ku. YPF1 and Ku also appear to bind DNA similarly. Southwestern blot experiments demonstrate that, like the Ku protein, the smaller YPF1 subunit binds DNA in the absence of the larger subunit. Further, cross-linking experiments indicate that, once again like the Ku protein, both subunits make contact with DNA when YPF1 binds as a heterodimer. YPF1 beta transcripts occur at low levels in all stages of Drosophila development except during oogenesis and early embryogenesis when they increase 25-fold. In situ hybridization localizes the beta gene to position 34C on the left arm of chromosome 2.

Sex-specific transcriptional regulation by the male and female doublesex proteins of Drosophila.

The somatic sexual phenotype of Drosophila is regulated by the sexual differentiation pathway. Male (DSXM) and female (DSXF) proteins encoded by doublesex (dsx), a gene at the end of this pathway, bind to three sites within a 127-bp enhancer that directs sex- and tissue-specific transcription of Yolk protein genes. We describe mutagenesis of these binding sites and the resulting effects on DSXM and DSXF binding in vitro and on gene regulation in wild-type and dsx mutant flies. The results demonstrate that DSXM represses and DSXF activates transcription from the two strongest binding sites. Thus, the pathway regulates sex-specific transcription through the male and female dsx proteins that act directly on the target gene, but with opposite effects.

The doublesex proteins of Drosophila melanogaster bind directly to a sex-specific yolk protein gene enhancer.

The doublesex (dsx) gene of Drosophila melanogaster encodes both male-specific and female-specific polypeptides, whose synthesis is regulated by alternative sex-specific splicing of the primary dsx transcript. The alternative splicing of the dsx mRNA is the last known step in a cascade of regulatory gene interactions that involves both transcriptional and post-transcriptional mechanisms. Genetic studies have shown that the products of the dsx locus are required for correct somatic sexual differentiation of both sexes, and have suggested that each dsx product functions by repressing expression of terminal differentiation genes specific to the opposite sex. However, these studies have not shown whether the dsx gene products function directly to regulate the expression of target genes, or indirectly through another regulatory gene. We report here that the male- and female-specific DSX proteins, expressed in E.coli, bind directly and specifically in vitro to three DNA sequences located in an enhancer region that regulates female-specific expression of two target genes, the yolk protein genes 1 and 2. This result suggests strongly that dsx is a final regulatory gene in the hierarchy of regulatory genes controlling somatic sexual differentiation.

Ovarian follicle cell enhancers from the Drosophila yolk protein genes: different segments of one enhancer have different cell-type specificities that interact to give normal expression.

This paper examines ovarian transcription of the divergently oriented yolk protein genes 1 and 2 (yp1 and yp2) of Drosophila melanogaster. We report germ line transformation results demonstrating that yp1 and yp2 are transcribed in the same subpopulations of ovarian follicle cells. Our results show that this expression pattern is directed by two enhancers: ovarian enhancer 1, located between the genes, and ovarian enhancer 2, located within the first exon of yp2. Analysis of the expression pattern resulting from alterations in ovarian enhancer 1 demonstrates that different segments of this enhancer have different positive or negative effects on the cell-type specificity of transcription.

DNA regions that regulate the ovarian transcriptional specificity of Drosophila yolk protein genes.

Yolk protein genes 1 and 2 (yp1 and yp2) of Drosophila melanogaster are divergently transcribed neighboring genes. Both are transcribed in only two tissues, the ovarian follicle cells and the fat bodies of adult females. Previous work has identified a yolk protein enhancer between the genes that is sufficient to direct transcription in one of the tissues, female fat bodies. Using germ-line transformation methods, we identify two cis-acting regions with positive effects on transcription in ovaries. One, a 301-bp region located between the genes, influences both genes and is an enhancer determining the stage and cell type specificity of ovarian transcription. The other, a 105-bp region located in the first exon of yp2, acts across the yp2 promoter region to stimulate yp1 transcription in ovaries. Additional observations suggest how a single enhancer influences both promoters.

The promoter region of the Drosophila alpha 2-tubulin gene directs testicular and neural specific expression.

The temporal and spatial expression pattern of the Drosophila melanogaster alpha 2-tubulin gene (alpha 2) has been investigated by examining the expression of an alpha 2-lacZ fusion gene. When this fusion gene is introduced into the germ line by P-element mediated transformation, expression is only detected in chordotonal organs and testes. Chordotonal organs, which are sensory organs of the peripheral nervous system, express the gene from late embryonic through adult stages in both males and females. Testicular expression occurs from larval through adult stages and is limited to germ-line cells, the primary and secondary spermatocytes and perhaps the early spermatids.

Identification of yolk protein factor 1, a sequence-specific DNA-binding protein from Drosophila melanogaster.

Transcription of the three yolk protein genes of Drosophila melanogaster is under strict developmental regulation. Understanding the mechanism of this regulation requires examination of the DNA sequences and protein factors necessary for normal transcriptional control. We have identified a sequence-specific DNA-binding protein, yolk protein factor 1 (YPF1), that has high affinity for a 31-bp sequence in the yolk protein 1 gene. This sequence is within the translated region at a site beginning 148 bp downstream of the transcription initiation site. DNA deletion and substitution analysis demonstrated that this sequence is necessary and sufficient for DNA/YPF1 interaction in vitro and is necessary for normal steady state levels of yolk protein 1 gene RNA in vivo. YPF1 binding activity was detected in extracts from late stage egg chambers and early stage embryos but not from tissues that express yolk protein genes.

Purification and properties of yolk protein factor I, a sequence-specific DNA-binding protein from Drosophila melanogaster.

We report the purification and some of the biochemical properties of yolk protein factor I (YPF1). This protein binds to a specific site in the yolk protein 1 gene (yp1) of Drosophila melanogaster. YPF1 has been purified to 95% homogeneity and consists of a heterodimer of two subunits with molecular weights 85,000 and 69,000. The protein is highly asymmetric with a frictional ratio of 1.56 which leads to calculated dimensions of 510 x 51 A when modeled as a prolate ellipsoid of revolution. It binds the yp1 DNA site with a protein/DNA stoichiometry of 1:1. Binding to that site is essentially irreversible with a dissociation rate constant of koff less than or equal to 2 x 10(-7) s-1, which gives the complex a dissociation half-life of approximately 55 days. The measured apparent second order association rate constant is 4 x 10(8) M-1 s-1 resulting in a calculated equilibrium dissociation constant of KD less than or equal to 5 x 10(-16) M. YPF1 also has a 10(8) selectivity for the yp1 site over poly(dA).poly(dT) (KDapp = 2 x 10(-8) M(nucleotide].

The alpha-lytic protease gene of Lysobacter enzymogenes. The nucleotide sequence predicts a large prepro-peptide with homology to pro-peptides of other chymotrypsin-like enzymes.

alpha-Lytic protease is a 19.8-kDa protein secreted from the Gram-negative bacterium Lysobacter enzymogenes. We have cloned and sequenced the gene for this serine protease. The nucleotide sequence contains an open reading frame which codes for the 198-residue mature enzyme and a potential prepro-peptide, also of 198 residues. The COOH-terminal 49 residues of the pro-peptide are significantly homologous to the propeptides of Streptomyces griseus proteases A and B. We suggest that this pro-peptide region facilitates formation of the active enzyme. A region bridging the NH2-terminal pre- and pro-peptides is homologous to a maize inhibitor of serine proteases. We speculate that this region inhibits enzymatic activity of the prepro-enzyme.