Pieter C. Wensink

The isolation and characterization of Drosophila yolk protein genes

We have isolated recombinant DNA clones that contain the genomic sequences coding for the three most abundant proteins in Drosophila eggs, the yolk proteins (YP1, YP2 and YP3). The identity of these cloned genes was established by a two-step procedure. We used the genes to isolate complementary mRNA from total Drosophila RNA; we than showed the in vitro translation products of the isolated mRNAs to be the yolk proteins by comparing their protease digestion products to those of yolk proteins isolated from eggs. An examination of these isolated genes and of their DNA complements in the Drosophila genome showed that each of the three coordinately expressed yolk proteins is encoded by a different single-copy gene. Three genes were cloned; each has a different pattern of restriction endonuclease sites and each appears to be homologous to a different yolk protein mRNA. Southern transfer blots demonstrated that there is only one copy of each gene in the Drosophila genome. Our structural studies have shown that these three genes are not adjacent. In situ hybridization to polytene chromosomes demonstrated, in fact, that the YP3 gene is approximately 1000 kb from the closely spaced YP1 and YP2 genes. Thus, if the coordinate synthesis of the yolk proteins is due to transcriptional control, there must be coordinate control of initiation at two distant sites.

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.

A tissue-specific transcription enhancer from the drosophila yolk protein 1 gene

The yolk protein 1 gene (yp1) of Drosophila melanogaster is expressed only in the ovarian follicle cells and the fat bodies of adult females. We have previously shown that a different cis-acting DNA region is required for each of these tissue specificities. In this paper we use germ line transformation to localize and characterized one of these tissue-specific regulatory regions. We demonstrate that a 125 bp segment of DNA located 196 bp upstream of the yp1 cap site is sufficient to determine the sex-, stage-, and fat body-specific expression of the yp1 gene. We also find that this region can confer yp1-specific expression on a heterologous Drosophila promoter. This specificity is retained when the region is in different orientations and at different distances from the heterologous promoter. Thus a small regulatory region acts in vivo as a positive enhancer to determine the fat body expression pattern of yp1.

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 inEscherichia 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.

The clustered and scrambled arrangement of moderately repetitive elements in drosophila DNA

An examination of cloned Drosophila DNA has revealed large clusters of densely spaced, short (less than or equal to 1 kb), moderately repetitive elements. Different clusters have many of the same repetitive elements, but these elements are arranged differently in each cluster. It is improbable that this clustered arrangement can be detected by conventional reassociation kinetic and electron microscopic techniques, but it can be detected and features of its fine structure can be determined by a two-dimensional version of Southerns blotting technique. The genomic organization of these clustered repetitive elements was investigated by hybridizing restriction fragments of cloned DNA to polytene chromosomes, to filter-bound recombinant DNA clones and to Southern blots of total Drosophila DNA. These studies demonstrated that clusters occur in euchromatic regions of the chromosomes and that at least one of the clusters has the same repetitive element organization in cloned and in chromosomal DNA. These studies also demonstrated that copies of the elements from one cluster are scattered in at least 1000 chromosomal regions. These regions appear to have differing concentrations of repetitive DNA, but together they account for a large fraction of Drosophilas moderately repetitive DNA. Aside from indicating the genomic organization of cluster elements, this work has identified cluster elements throughout a 9 kb region neighboring one of the heat shock genes, throughout the intron of the major rDNA repeat and within the apparently transposable element, 412.

Developmental regulation of Drosophila α-tubulin genes

Abstract Transcripts from the four different Drosophila melanogaster α-tubulin genes were detected by filter hybridization experiments that used subcloned fragments from each gene as hybridization probes. These hybridization experiments demonstrated that each gene is transcribed. All of the transcripts are found on polysomes and are long enough to encode an α-tubulin protein. The hybridization studies were extended to examine the developmental pattern of RNA concentrations. The concentration of RNAs from the α2 and α4 genes vary independently and dramatically, while those from α1 and α3 have parallel variations. We conclude that at the RNA level of expression, two α-tubulin genes are regulated in parallel and two genes are not. We hypothesize that the different concentration patterns reflect different functions for the protein products of each gene.

Sequence and structure conservation in yolk proteins and their genes

The yp1 and yp2 genes of Drosophila code for egg yolk proteins. Their transcription is hormone-dependent and co-ordinate. In this paper we describe the complete nucleotide sequence of the yp2 gene and of the region between these two divergently transcribed genes. We also map the mature messenger RNA on the yp2 gene sequence. We then use this information and similar information previously determined for the yp1 gene to find features common to the two genes and to the two proteins. Most features common to the nucleotide sequences flanking the two genes are consensus sequences that have been found in many other eukaryotic genes. An unusual feature common to the two genes is a potential stem-and-loop structure, which has a TATA box, a capping site, ribosomal RNA homology and then a translation initiation codon at the four successive junctions between single strands and duplexes. A second unusual common feature is a 13-nucleotide sequence that is similar to a recently proposed consensus sequence for the progesterone-receptor binding site. We speculate that the stem-and-loop structure and the conserved 13-nucleotide sequence may be involved in the co-ordinate, hormone-dependent expression of the genes. In examining features common to the two proteins we find that the sequences are 53% homologous and the predicted secondary structures are nearly identical. We propose that the conserved secondary structure is important to the oligomerization and perhaps to other activities common to these proteins.

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.

Isolation and analysis of recombinant DNA molecules containing yeast DNA.

2500 recombinant plasmids containing insertions of yeast nuclear DNA have been cloned in Escherichia coli. It can be calculated that about 85% of the yeast genome is represented in this collection. The clones have been characterized by hybridization to purified RNA species. Of the 2000 clones examined, 75 contain insertions of yeast ribosomal DNA, 201 contain insertions of yeast tRNA genes, and 26 contain DNA sequences that are complementary to abundant mRNA species.

α-tubulin genes of drosophila

Abstract Four Drosophila α-tubulin genes have been isolated on recombinant DNA molecules. The identity of two of these genes (Tα1 and Tα2) was established by isolating complementary mRNAs and then examining the in vitro translation products of the mRNAs. The one- and two-dimensional gel patterns and the peptide maps of the in vitro products were indistinguishable from those of embryonic α-tubulin. In turn, the embryonic tubulin was identified by determining its amino-terminal sequence. We identified two other cloned α-tubulin genes (Tα3 and Tα4) by their complementarity to Tα1 and Tα2. Maps of restriction endonuclease sites indicate that the four genes are different. DNA hybridization studies demonstrated, however, that three of them have extensive sequence homology with each other and slight homology with the fourth, Tα4. Hybridization to genomic DNA fragments indicated that the four cloned genes account for all of the different α-tubulin genes of Drosophila melanogaster. Three of them are present only once in the haploid genome; the other, Tα1, is present in either one or two copies. Each of the four genes hybridizes in situ to a different site on the third chromosome (67C4-6, 84B3-C8, 84D5-8 and 85E6-15).