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.

Mobile element insertions causing mutations in the Drosophila suppressor of sable locus occur in DNase I hypersensitive subregions of 5'-transcribed nontranslated sequences.

The locations of 16 mobile element insertions causing mutations at the Drosophila suppressor of sable [su(s)] locus were determined by restriction mapping and DNA sequencing of the junction sites. The transposons causing the mutations are: P element (5 alleles), gypsy (3 alleles), 17.6, HMS Beagle, springer, Delta 88, prygun, Stalker, and a new mobile element which was named roamer (2 alleles). Four P element insertions occur in 5' nontranslated leader sequences, while the fifth P element and all 11 non-P elements inserted into the 2053 nucleotide, 5'-most intron that is spliced from the 5' nontranslated leader approximately 100 nucleotides upstream of the translation start. Fifteen of the 16 mobile elements inserted within a approximately 1900 nucleotide region that contains seven 100-200-nucleotide long DNase I-hypersensitive subregions that alternate with DNase I-resistant intervals of similar lengths. The locations of these 15 insertion sites correlate well with the roughly estimated locations of five of the DNase I-hypersensitive subregions. These findings suggest that the features of chromatin structure that accompany gene activation may also make the DNA susceptible to insertion of mobile elements.

Molecular and genetic organization of the suppressor of sable and minute (1) 1B region in Drosophila melanogaster.

Recessive mutations at the suppressor of sable [su(s)] locus in Drosophila melanogaster result in suppression of second site mutations caused by insertions of the mobile element 412. In order to determine whether su(s) mutations might have other phenotypes, a saturation mapping of the su(s) region was carried out. The screen yielded 76 mutations that comprise ten genetic complementation groups ordered distal to proximal as follows: l(1)1Bh, l(1)1Bi, M(1)1B, su(s), l(1)1Bk, l(1)1Ca, mul, tw, l(1)lDa and brc. Twenty-three of the mutations are su(s) alleles, and all are suppressors of the 412-insertion-caused v1 allele. Although the screen could have detected su(s) mutations causing sex-specific dominant lethality or sterility as well as all types of recessive lethality or sterility, the only other phenotype observed was male sterility that is enhanced by cold temperature. This type of sterility is exhibited only by alleles induced by base-substitution-causing mutagens. Genetic functions of the poly(A+) messages transcribed from the su(s) microregion were identified by the reintroduction of cloned sequences into embryos by P element transformation. su(s) function has been attributed to a 5-kb message. The segment of DNA encoding only this 5-kb message rescues both the suppression and cold-sensitive male sterility phenotypes of su(s). Minute (1) 1B has been provisionally identified as encoding a 3.5-kb message; lethal (1)1Bi encodes a 1-kb message; and lethal (1)1Bk encodes a 4-kb message. The possible functions of su(s) and M(1)1B are discussed.

Sites of P element insertion and structures of P element deletions in the 5' region of Drosophila melanogaster RpII215.

Several P element insertion and deletion mutations near the 5' end of Drosophila melanogaster RpII215 have been examined by nucleotide sequencing. Two different sites of P element insertion, approximately 90 nucleotides apart, have been detected in this region of the gene. Therefore, including an additional site of P element insertion within the coding region, there are at least three distinct sites of P element insertion at RpII215. Both 5' sites are within a noncoding portion of transcribed sequences. The sequences of four revertants of one P element insertion mutation (D50) indicate that the P element is either precisely deleted or internally deleted to restore RpII215 activity. Partial internal deletions of the P element result in different RpII215 activity levels, which appear to depend on the specific sequences that remain after excision.

Molecular cloning of suppressor of sable, a Drosophila melanogaster transposon-mediated suppressor.

A hybrid dysgenesis-induced allele [su(s)w20] associated with a P-element insertion was used to clone sequences from the su(s) region of Drosophila melanogaster by means of the transposon-tagging technique. Cloned sequences were used to probe restriction enzyme-digested DNAs from 22 other su(s) mutations. None of three X-ray-induced or six ethyl methanesulfonate-induced su(s) mutations possessed detectable variation. Seven spontaneous, four hybrid dysgenesis-induced, and two DNA transformation-induced mutations were associated with insertions within 2.0 kilobases (kb) of the su(s)w20 P-element insertion site. When the region of DNA that included the mutational insertions was used to probe poly(A)+ RNAs, a 5-kb message was detected in wild-type RNA that was present in greatly reduced amounts in two su(s) mutations. By using strand-specific probes, the direction of transcription of the 5-kb message was determined. The mutational insertions lie in DNA sequences near the 5' end of the 5-kb message. Three of the seven spontaneous su(s) mutations are associated with gypsy insertions, but they are not suppressible by su(Hw).

Molecular characterization of the Drosophila vermilion locus and its suppressible alleles.

We have cloned vermilion (v), one of the genes required for brown eye pigment synthesis in Drosophila, using a mutant (vH2a) "tagged" with the transposon P factor. Mutations that disrupt v gene expression are clustered within approximately 2 kilobases of DNA. A 1.4-kilobase transcript, homologous to this same region, is present in v+ RNA but absent in RNA from several v mutants. The spontaneous v alleles that are suppressed by the suppressor of sable [su(s)] are apparently identical insertions of 412, a copia-like transposable element. Preliminary evidence suggests that su(s)-suppressible alleles at other loci may also be 412 insertions.

Characterization of Allozyme Null and Low Activity Alleles from Two Natural Populations of DROSOPHILA MELANOGASTER.

Null and low enzyme activity alleles recovered from two natural populations were analyzed for a number of genetic and biochemical properties. Analysis of 58 mutations at 14 loci showed that all but one allele were genetically viable and fertile, four alleles were associated with chromosome rearrangements, 28 alleles retained some enzyme activity, 13 alleles formed an active heterodimer with active alleles and five alleles showed partial interallelic complementation. Available evidence indicates that this sample includes mutations resulting from lesions in both coding and regulatory sequences. Certain mutations may be caused by transposable element insertions.

Frequent Imprecise Excision among Reversions of a P Element-Caused Lethal Mutation in Drosophila.

RpII215(D) (50) (= D50) is a lethal mutation caused by the insertion of a 1.3-kb P element 5' to sequences encoding the largest (215 kilodaltons) subunit of Drosophila RNA polymerase II. In dysgenic males D50 reverted to nonlethality at frequencies ranging from 2.6 to 6.5%. These reversions resulted from loss of P element sequences. Genetic tests of function and restriction enzyme analysis of revertant DNAs revealed that 35% or more of the reversion events were imprecise excisions. Two meiotic mutations that perturb excision repair and postreplication repair (mei-9(a) and mei-41(D5), respectively) had no influence on reversion frequency but may have increased the proportion of imprecise excisions. We suggest that these excisions are by-products of, rather than intermediates in, the transposition process.

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.

Estimation of genetic variability in natural populations of Drosophila simulans by two-dimensional and starch gel electrophoresis.

Genic variation in natural populations of Drosophila simulans was surveyed using allozymic and two-dimensional electrophoretic techniques. Consistent with some previous reports, allozymic heterozygosity appeared lower than in the sibling species D. melanogaster (0.07 vs. 0.16). No variation was detected by two-dimensional electrophoresis of 19 lines scored for 70 abundant proteins. This is consistent with reported reductions in estimates of genic heterozygosity by two-dimensional electrophoresis in D. melanogaster, Mus musculus, and man. Although the amount of intraspecific variation detected in abundant proteins was lower than that detected for allozymes in D. simulans and D. melanogaster, the genetic distances between the sibling species calculated from the two data sets are not significantly different (0.35 and 0.20). The allozyme and two-dimensional electrophoresis data confirmed the impression from other measures of genetic variation (mitochondrial DNA restriction maps and inversion polymorphisms) that D. simulans is substantially less variable than D. melanogaster.

Null allele frequencies at allozyme loci in natural populations of Drosophila melanogaster.

We have sampled a London population of Drosophila melanogaster for null alleles at twenty-five allozyme loci. The same loci and biochemical techniques were used as in our previous survey of a North Carolina population (Voelker et al. 1980). This second survey is completely concordant with the first. No nulls were detected among the five X-linked loci. The mean frequency of nulls at the twenty autosomal loci was 0.0023. Although there is significant interlocus heterogeneity, the two populations appear to have the same frequencies at each locus. This suggests that null alleles at these allozyme loci are in mutation-selection balance, and we estimate the average heterozygous effect of an allozyme null to be 0.0015. Consideration of allozyme null-allele frequencies, the effects of allozyme null alleles on viability and fertility and the generally greater amount of genetic variability at allozyme loci determined by electrophoresis lead us to doubt the validity of generalizing from allozyme data to the whole genome.

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.

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 87 F12-14 and 88 C1-3 and between 87 B5-6 and 87 B8-10, 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 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.

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.

Spontaneous Allozyme Mutations in DROSOPHILA MELANOGASTER: Rate of Occurrence and Nature of the Mutants.

After additional generations of accumulation of allozyme mutants, the 1,000 lines of Mukai and Cockerham (1977) were again screened for the same five loci (alpha-Gpdh, cMdh, Adh, Hex-C and alpha-Amy), as well as for two new loci (Got-2 and Dip-A). Based on 3,111,598 allele generations: (1) the average mutation rate to new mobility variants with normal function was estimated to be 1.28 x 10(-6), and (2) the average mutation rate to null alleles was estimated to be 3.86 x 10(-6). A qualitative analysis of the nulls provided evidence that most of the mutants recovered are due to base substitutions. No apparent correlation was observed between structural gene size and mutation rate.

Enzyme null alleles in natural populations of Drosophila melanogaster: Frequencies in a North Carolina population.

A Raleigh, NC, population of Drosophila melanogaster was sampled for the presence of enzyme null alleles at 25 loci. No nulls were found at any of five X-linked loci. Nulls were recovered at 13 of 20 autosomal loci; the weighted mean frequency for all 20 autosomal loci was estimated to be 0.0025. A consideration of the effects of these null alleles on viability strongly suggests that, although they may contribute to so-called polygenic variation, they are not representative of the entire genome.

Enzyme mutants induced by low-dose-rate gamma-irradiation in Drosophila: frequency and characterization.

One thousand lines of a balanced lethal strain of Drosophila melanogaster heterozygous at seven allozyme loci were subjected to chronic (0.15 rad/min) gamma-irradiation (137Cs). After 15 generations of exposure they were screened by gel electrophoresis for newly arisen null mutants and/or mobility variants. Seven independent nulls were recovered. 1) None were associated with a cytogenetically detectable aberration; 2) one of six analyzed was associated with recessive lethality, suggesting association with a submicroscopic interlocus deficiency; 3) three of five which could be analyzed for cross-reacting material (CRM) production were CRM-positive; and 4) two of six produced an active heterodimer with an active normal allele. The results taken together suggest that most of the mutations are point mutations rather than small deficiencies.

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 (mMdh) found at 62.6 on the third chromosome and included in Df(3R)R14, 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-gamma 10069/Df(2L)J-der-27) were both viable and fertile.

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 locataion of Pgm, the structural gene for phosphoglucomutase which has been located genetically at 3-43.4, was determined to be in 72D1-5.