Robert A. Voelker

Molecular cloning of sequences from a Drosophila RNA polymerase II locus by P element transposon tagging.

We have identified a lethal mutation in the D. melanogaster RNA polymerase II locus, RpIIC4, caused by insertion of a transposable element associated with the phenomenon of hybrid dysgenesis (P element). Using previously cloned P element sequences as a hybridization probe we have isolated a hybrid lambda phage clone carrying a 10 kb genomic DNA fragment containing a 1.3 kb P element insert and flanking sequences from the RpII locus. The non-P sequences in this clone (lambda DmRpII-1) hybridize to polytene chromosome band region 10C, the cytogenetic location of RpIIC4, and revertants which lose the lethal RNA polymerase II mutation also lose P element sequences from the locus. We have generated several additional P element insertions into the locus and shown that they can occur at two or more different sites. These experiments illustrate that mutagenesis by P element insertion and use of cloned P DNA to retrieve the DNA sequences into which insertion has occurred may be a general method for cloning genetically defined loci in Drosophila.

Genetic and biochemical characterization of mutants at an RNA polymerase II locus in D. melanogaster

We previously described an alpha-amanitin-resistant mutant of D. melanogaster (AmaC4 or simply C4) with an altered, amanitin-resistant RNA polymerase II. We have now more fully characterized this mutant genetically and biochemically. We genetically mapped C4 to position 35.66 on the X chromosome and cytogenetically localized it to the polytene chromosome band interval 10C2-10D4. We then demonstrated that C4 is allelic to a previously known lethal-mutable locus I(1)L5 in this chromosomal region. Several known lethal alleles of L5 in fact affected the properties of RNA polymerase II in vitro. Following EMS mutagenesis of the C4-bearing chromosome we recovered new lethal L5 alleles, some of which were shown biochemically to have an altered amanitin-resistance polymerase II component. Furthermore, we induced mutants of C4 that had lost amanitin-resistance and showed that these mutants were also lethal alleles of L5. All the lethal alleles of L5 failed to completely complement each other genetically, and when analyzed biochemically their polymerase II displayed altered enzymatic properties. We conclude that C4 is an allele of the L5 locus and that this locus is most probably a structural gene for a subunit of RNA polymerase II. Some of the mutants at this locus display developmental abnormalities.

The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins.

The nucleotide sequence of the Drosophila melanogaster suppressor of sable [su(s)] gene has been determined. Comparison of genomic and cDNA sequences indicates that an approximately 7,860-nucleotide primary transcript is processed into an approximately 5-kb message, expressed during all stages of the life cycle, that contains an open reading frame capable of encoding a 1,322-amino-acid protein of approximately 150 kDa. The putative protein contains an RNA recognition motif-like region and a highly charged arginine-, lysine-, serine-, aspartic or glutamic acid-rich region that is similar to a region contained in several RNA-processing proteins. In vitro translation of in vitro-transcribed RNA from a complete cDNA yields a product whose size agrees with the size predicted by the open reading frame. Antisera against su(s) fusion proteins recognize the in vitro-translated protein and detect a protein of identical size in the nuclear fractions from tissue culture cells and embryos. The protein is also present in smaller amounts in cytoplasmic fractions of embryos. That the su(s) protein has regions similar in structure to RNA-processing protein is consistent with its known role in affecting the transcript levels of those alleles that it suppresses.

Genetic and cytogenetic studies of four glycolytic enzymes in Drosophila melanogaster: Aldolase, triosephosphate isomerase, 3-phosphoglycerate kinase, and phosphoglucomutase

Four glycolytic enzymes in Drosophila melanogaster have been genetically and/or cytogenetically mapped. The structural gene for aldolase (Ald) has been genetically mapped to 3-91.5 and cytogenetically localized to 97A-B. Tpi, the structural gene for triosephosphate isomerase, has been genetically mapped to 3-101.3 and cytogenetically localized to 99B-E. Utilizing closer-flanking markers than the previous mapping, Pgk, the structural gene for 3-phosphoglycerate kinase, has been mapped to 2-5.9; cytogenetically it was found to lie in the interval between 22D and 23E3. The cytogenetic location of Pgm, the structural gene for phosphoglucomutase which has been located genetically at 3-43.4, was determined to be in 72D1-5.

Genetic and cytogenetic studies of malic enzyme in Drosophila melanogaster

The genetic and cytogenetic locations of the structural gene (Men) for malic enzyme have been determined. Men maps genetically between kar and ry at 51.73±0.02. Cytogenetically, Men probably lies in the proximal edge of 87D1,2, based on the results of mapping utilizing a number of deficiencies with breakpoints in that region. A number of null alleles have been recovered; heterozygotes for these nulls and a Men deficiency are both viable and fertile. These findings are related to the one band, one functional unit model of salivary gland chromosome structure.

Genetic and molecular variation in the RpII215 region of Drosophila melanogaster

SummaryThe RpII215 region of the X chromosome of Drosophila melanogaster was investigated to identify genetic functions and correlate these with the known molecular organization of the region. Five genetic loci were identified in a subregion that is reported to transcribe nine or more messages. One locus is nod, which causes meiotic abnormalities, and three other loci are recessive lethal mutations whose developmental lesions are unknown. The fifth and most mutable of the loci is RpII215, which encodes the 215,000 dalton subunit of RNA polymerase II. Mutant effects of RpII215 alleles include: temperature-dependent (heat and cold) survival, altered sensitivity to α-amanitin, male sterility, maternal effects and epistatic enhancement of mutant effects of other loci.

Comparative studies of allozyme loci in Drosophila simulans and Drosophila melanogaster. I. Three dipeptidase loci.

Genetic variation at three dipeptidase loci (Dip-A, Dip-B, and Dip-C) in Drosophila simulans was analyzed by starch gel electrophoresis. Dip-A was found to be polymorphic in four populations, while Dip-B and Dip-C were found to be polymorphic in one. The numbers of different alleles found at each respective locus were: Dip-A, two; Dip-B, two; and Dip-C, three. Dip-A was genetically mapped at 57.9 on the second chromosome, and Dip-B and Dip-C at 80.9 and 87.9 on the third chromosome, respectively. Neither Dip-B nor Dip-C has been mapped in D. melanogaster because both loci are apparently monomorphic. Their map positions in D. simulans with respect to flanking markers whose homologous genes have been cytogenetically localized in D. melanogaster suggested that they might be mapped cytogenetically by using available deficiencies in D. melanogaster. Accordingly, by the construction of interspecific hybrids which carried deficiencies of melanogaster and an allele of simulans with a mobility different from that of the fixed melanogaster allele, Dip-B and Dip-C were localized between 87F12-14 and 88C1-3 and between 87B5-6 and 87B8-10, respectively, in the salivary gland chromosomes of D. melanogaster. The similarity between these two species is discussed on the basis of these findings.

Genetic and cytogenetic studies of the malate dehydrogenases of Drosophila melanogaster.

Genetic and cytogenetic locations of the structural genes for the NAD-dependent malate dehydrogenases have been studied. The mitochondrial form (mMDH) is coded for by a gene (Mdh) found at 62.6 on the third chromosome and included in Df(3R)P14, which includes 90C2–91A3 in the salivary gland chromosomes. Based on its inclusion within several J (Jammed; 2–41.0) deficiencies, the structural gene (cMdh) for the cytoplasmic form (cMDH) was determined to lie in region 31B-E, confirming the earlier finding of Grell. Flies lacking any cMDH activity (cMdhn-γ10069/Df(2L)J-der-27) were both viable and fertile.

Genetic and biochemical studies on glutamate-pyruvate transaminase from Drosophila melanogaster

We have used electrophoretic variants of glutamate-pyruvate transaminase (GPT, E.C. 2.6.1.2) in Drosophila melanogaster to genetically map the structural gene to position 42.6 on the X chromosome. By pseudodominance tests over several deficiencies we have localized it cytogenetically to the interval 11Fl-2 to 12Al-2. The sedimentation constant (s20,w) of the native enzyme was determined in sucrose density gradients to be 5.9 and the native molecular weight approximately 87,000. The similarity in physical properties to mammalian enzymes suggests that the enzyme may also be dimeric in D. melanogaster.