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M M Green

Publications and source records attributed to M M Green.

At least 55 records · Page 3Linked to original sources
PubMed1989

Isolation of a cDNA encoding functional Drosophila alcohol dehydrogenase in Escherichia coli and purification of the bacterially produced enzyme.

Because of the severe limitations on growing large quantities of Drosophila affinidisjuncta in the laboratory, direct purification of alcohol dehydrogenase (ADH) from this species has proven impossible. As an alternative source of this enzyme, a cDNA encoding functional ADH was isolated from a newly constructed cDNA library made from larval poly(A)-containing RNA. The cDNA was recovered by virtue of its hybridization to a previously isolated genomic ADH gene. Nucleotide sequence analysis confirmed the identity of the newly isolated cDNA. When the cDNA was inserted in the proper orientation downstream of the lac promoter on the vector pUC8, the cDNA directed the synthesis of functional ADH by the bacterial host. The bacterially produced enzyme was purified to homogeneity and used to elicit polyclonal antibodies in rabbits. The purified ADH has identical apparent subunit molecular weight to that of authentic ADH in larval fly extracts as determined by immunoblotting. Further, comparisons of the kinetic parameters of the bacterially produced enzyme and ADH activity in larval fly extracts indicate similar substrate preferences, pH dependencies, and Km values for 2-propanol and NAD. These results show that expression of a cDNA in Escherichia coli is a valid strategy for isolation of an ADH that would otherwise be difficult or impossible to purify.

Alcohol Dehydrogenase↗
PubMed1988

Reversion of a gypsy-induced mutation at the yellow (y) locus of Drosophila melanogaster is associated with the insertion of a newly defined transposable element.

To understand the molecular basis of the phenotype of gypsy-induced mutations, we have analyzed the structure of phenotypic revertants of the y2 allele, which is caused by the insertion of the gypsy element into the 5' region of the yellow (y) locus. Seven spontaneous revertants examined fall into two different classes. Three of these revertants arose by homologous recombination between the two gypsy long terminal repeats (LTRs), leaving behind a solo LTR. Four additional revertants contain an intact 3' LTR and half of the 5' LTR, but the central portion of gypsy has been replaced by a different 6.5-kilobase transposable element that contains a poly(A) tail. These results suggest that the mutagenic effect of the gypsy element is not due to its insertion into sequences necessary for transcription or to the distancing between the yellow promoter and remote regulatory sequences but is a consequence of idiosyncratic properties of the element itself.

Animals↗
PubMed1988

Genetic instability in Drosophila melanogaster: P-element mutagenesis by gene conversion.

We report the molecular characterization of several P element-induced mutations and their revertants at the yellow (y) locus of Drosophila melanogaster. One of the mutants analyzed, y76d28, results from the insertion of a P element into the 5'-transcribed, untranslated portion of the y gene. Sequence analysis of several revertants of y76d28 shows that P excision occurs imprecisely. These events result in insertion of additional ATG codons in the y locus mRNA but are without phenotypic effect. In addition, we describe the molecular structure of P-associated mutations induced in a near wild-type revertant of y76d28 that carries an internally deleted 0.4-kilobase P element in the 5' noncoding region. Sequence analysis of two of these mutants demonstrates that they arose as a result of the integration of a larger P element at the exact location as in the parental stock without the 8-base-pair additional duplication associated with P insertions. The phenotype of these y alleles is dependent on the size and orientation of the integrated P element. We infer that P-element replacement in these mutants has occurred by a recombination/gene conversion mechanism.

Alleles↗
PubMed1988

Mutant gene phenotypes mediated by a Drosophila melanogaster retrotransposon require sequences homologous to mammalian enhancers.

We have analyzed the molecular structure of phenotypic revertants of gypsy-induced mutations to understand the molecular mechanisms by which this retrotransposon causes mutant phenotypes in Drosophila melanogaster. The independent partial revertants analyzed are caused by the insertion of different transposons into the same region of gypsy. One partial revertant of the yellow allele y2 arose as a consequence of the insertion of the jockey mobile element into gypsy sequences, whereas a second incomplete revertant is due to the insertion of the hobo transposon. In addition, a previously isolated partial revertant of the Hairy-wing allele Hw1 resulted from the integration of the BS transposable element into the same gypsy sequences. The region affected by the insertion of the three transposons contains 12 copies of a repeated motif that shows striking homology to mammalian transcriptional enhancers. Our results suggest that these sequences, which might be involved in the transcriptional control of the gypsy element, are also responsible for the induction of mutant phenotypes by this retrotransposon.

Alleles↗
PubMed1988

Partial revertants of the transposable element-associated suppressible allele white-apricot in Drosophila melanogaster: structures and responsiveness to genetic modifiers.

The eye color phenotype of white-apricot (wa), a mutant allele of the white locus caused by the insertion of the transposable element copia into a small intron, is suppressed by the extragenic suppressor suppressor-of-white-apricot (su(wa] and enhanced by the extragenic enhancers suppressor-of-forked su(f] and Enhancer-of-white-apricot (E(wa]. Derivatives of wa have been analyzed molecularly and genetically in order to correlate the structure of these derivatives with their response to modifiers. Derivatives in which the copia element is replaced precisely by a solo long terminal repeat (sLTR) were generated in vitro and returned to the germline by P-element mediated transformation; flies carrying this allele within a P transposon show a nearly wild-type phenotype and no response to either su(f) or su(wa). In addition, eleven partial phenotypic revertants of wa were analyzed. Of these, one appears to be a duplication of a large region which includes wa, three are new alleles of su(wa), two are sLTR derivatives whose properties confirm results obtained using transformation, and five are secondary insertions into the copia element within wa. One of these, waR84h, differs from wa by the insertion of the most 3' 83 nucleotides of the I factor. The five insertion derivatives show a variety of phenotypes and modes of interaction with su[f) and su(wa). The eye pigmentation of waR84h is affected by su(f) and E(wa), but not su(wa). These results demonstrate that copia (as opposed to the interruption of white sequences) is essential for the wa phenotype and its response to genetic modifiers, and that there are multiple mechanisms for the alteration of the wa phenotype by modifiers.

Alleles↗
PubMed1987

The spontaneous mutation rate in Drosophila simulans.

The purpose of this investigation was to determine whether there exists a correlation in Drosophila between the spontaneous mutation rate and the amount of dispersed middle repetitive (mobile) DNA sequences. The amount of these sequences is 7 times less in Drosophila simulans as compared to Drosophila melanogaster. Therefore, if a correlation exists, the spontaneous mutation rate in Drosophila simulans should be 7 times lower than that in Drosophila melanogaster. We isolated an X-chromosome inversion after X-irradiation of wild-type Drosophila simulans males, that reduced crossing-over between white and forked, two X-linked visible markers, to less than 1%. This inversion was subsequently used to determine the sex-linked recessive lethal mutation rate in Drosophila simulans males of a laboratory strain marked with white. The frequency of these lethal mutations found is not different from that observed in Drosophila melanogaster males of laboratory strains.

Animals↗
PubMed1987

Genetic instability in Drosophila melanogaster: cytogenetic analysis of MR-induced X-chromosome deficiencies.

We present data that demonstrate that three MR elements isolated from wild populations of Drosophila melanogaster on two continents can cause large deletions of the X chromosome in males. The deleted chromosomes, termed mini-X chromosomes, are induced at a frequency of approximately 1:4000 in chromosomes that are initially free of P elements. In situ hybridizations using a cloned P sequence as a probe fail to reveal any sequences homologous to the nomadic P family at the deletion breakpoints. Genetic analysis of 12 such mini-X chromosomes also reveals that there are no "hotspots" of chromosome breakage and that there must have been a minimum of three distinct distal breakpoints and five different proximal breakpoints in the formation of these deleted chromosomes. In fact all 12 proximal and 12 distal breakpoints may well be unique. Our data show that MR elements generate essentially random breaks along the X chromosome. We emphasize that we find no involvement of P sequences in the chromosome breakage process, consonant with the notion that MR elements exert their influence on processes involved in mitotic crossing-over.

Animals↗
PubMed1986

Genetic-molecular basis for a simple Drosophila melanogaster somatic system that detects environmental mutagens.

We have developed a simple, objectively scorable test for the mutagenicity of chemical compounds which can be fed Drosophila melanogaster. The test depends upon the somatic reversion of the X chromosome, recessive eye color mutation, white-ivory (wi) to wild type (w+). Reversions are scored as clones of w+ facets in the wi eyes of eclosing adults. To increase the sensitivity, a tandem quadruplication containing four wi mutations was synthesized. Thus, in homozygous females eight wi mutations are potentially revertible. Six mutagenic compounds, all alkylating agents, all gave positive results at several concentrations tested. Molecular analysis demonstrates that the induced reversions, germinal and somatic, are associated with the loss of 2.9-kilobase DNA duplicated in the wi mutation.

Animals↗
PubMed1986

Genetic instability in Drosophila melanogaster: the genetics of an MR element that makes complete P insertion mutations.

An MR element that maps to a specific locus in chromosome 3 of Drosophila melanogaster has the unusual feature of producing complete P (a mobile element) sequence mutations at several X chromosome loci. This MR also increases the frequency of mitotic recombination. Evidence is given for the transposition of MR. The complete P insertion mutations are autonomously unstable and are capable of causing otherwise stable incomplete (defective) P insertion mutations to revert. These results complement the analysis of P element functions with a synthetic complete P derivative. The genetic basis for the mutational-mitotic recombinational components of "hybrid dysgenesis" is conveniently explicable in terms of MR elements present in the genomes of flies present in the wild.

Animals↗
PubMed1984

The zeste-white interaction: induction and genetic analysis of a novel class of zeste alleles.

The recovery and analysis of a novel class of zeste mutations is described. z and z do not require two w genes for their expression unless the z gene is present. Analysis of genetic interactions among zeste alleles proved that z mutations are strong antagonists of the z gene product. z is readily reverted by X-rays or by ethylmethane sulphonate to a range of intermediate zeste alleles and thus it is considered to be an insertional mutation. We suggest that z and op mutations arose as a result of insertions in a presumptive control region, and two alternative models for the structure of the zeste locus are evaluated.

Journal Article↗
PubMed1984

mu-2: mutator gene in Drosophila that potentiates the induction of terminal deficiencies.

An x-ray-dependent mutator on chromosome 3 of Drosophila melanogaster is described that specifically increases the recovery of deletions for chromosomal tip regions. Such deficiencies can be induced on any chromosome. More centromere proximal mutations, as assayed by the sex-linked recessive lethal test, are not increased over the wild-type control. As far as can be determined by genetic, cytological, and molecular assays, the deletions extend to the very end of the chromosome involved. In addition, the frequency of these deletions is directly proportional to x-ray dose, suggesting that they are one-break rearrangements. It is proposed that the mutator is blocked in a major pathway for the repair of DNA double-strand breaks, and that a minor repair pathway is responsible for the addition of new telomeres under these conditions.

Animals↗
PubMed1983

Unequal crossing-over associated with asymmetrical synapsis between nomadic elements in the Drosophila melanogaster genome.

The molecular structure of reciprocal duplications and deficiencies produced by unequal crossing-over at the white (w) locus of Drosophila melanogaster females heterozygous for the alleles w(a) and w(a4) has been examined. A transposable, copia-like element is found at the rearrangement breakpoints. Further characterization indicates that asymmetrical pairing between two copies of this element, which are at least 60 kilobases apart in the parental chromosomes, followed by a crossover within the paired elements, is responsible for the duplication and deficiencies observed. The frequency of these events is high compared with normal homologous exchange, implying that synaptic pairing during meiosis must be sufficiently flexible as to allow efficient recognition of sequences located in nonidentical positions on homologous chromosomes. These results suggest a possible mechanism for the generation of tandem duplications in eukaryotic organisms.

Journal Article↗
PubMed1983

Special training under simulated stat lab conditions.

A course has been devised to simulate a hospital stat lab environment for students in a 2-year MLT Associate Degree program. This course ensures that each student will individually perform a wide variety of laboratory procedures and report results under hospital-like circumstances. This course, which has received favorable comment from NAACLS, circumvents problems of insufficient placement situations, inadequate supervision, and limited variety of student experience in hospital sites. Course procedures, objectives, and student evaluation methods are described.

Clinical Laboratory Techniques↗
PubMed1982

The mutagenic effects of diepoxybutane in wild-type and mutagen-sensitive mutants of Drosophila melanogaster.

Genetic tests reported here demonstrate that among the DEB-induced mutants on 2 X-chromosome loci, viz. y and w, at a minimum, one-third are chromosome deletions. Among 11 MMS-sensitive mutants tested, 9 are also somatically sensitive to DEB. In addition direct genetic tests established that the capacity to repair DEB damage induced in sperm is impaired in females homozygous for 2 mutagen-sensitive mutants. By inference the same is also the case in females homozygous for 3 other mutagen-sensitive mutants.

Animals↗