Analysis of Two Cosmid Clones from Chromosome 4 of Drosophila melanogaster Reveals Two New Genes Amid an Unusual Arrangement of Repeated Sequences

Chromosome 4 from Drosophila melanogaster has several unusual features that distinguish it from the other chromosomes. These include a diffuse appearance in salivary gland polytene chromosomes, an absence of recombination, and the variegated expression of P-element transgenes. As part of a larger project to understand these properties, we are assembling a physical map of this chromosome. Here we report the sequence of two cosmids representing ∼5% of the polytenized region. Both cosmid clones contain numerous repeated DNA sequences, as identified by cross hybridization with labeled genomic DNA, BLAST searches, and dot matrix analysis, which are positioned between and within the transcribed sequences. The repetitive sequences include three copies of the mobile element Hoppel, one copy of the mobile element HB, and 18 DINE repeats. DINE is a novel, short repeated sequence dispersed throughout both cosmid sequences. One cosmid includes the previously described cubitus interruptus(ci) gene and two new genes: that a gene with a predicted amino acid sequence similar to ribosomal protein S3a which is consistent with the Minute(4)101 locus thought to be in the region, and a novel member of the protein family that includes plexin and met–hepatocyte growth factor receptor. The other cosmid contains only the two short 5′-most exons from thezinc-finger-homolog-2 (zfh-2) gene. This is the first extensive sequence analysis of noncoding DNA from chromosome 4. The distribution of the various repeats suggests its organization is similar to the β-heterochromatic regions near the base of the major chromosome arms. Such a pattern may account for the diffuse banding of the polytene chromosome 4 and the variegation of many P-element transgenes on the chromosome.

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Identification of Chromosome Inheritance Modifiers in Drosophila melanogaster

Genetics ◽

10.1093/genetics/157.4.1623 ◽

2001 ◽

Vol 157 (4) ◽

pp. 1623-1637 ◽

Cited By ~ 7

Author(s):

Kenneth W Dobie ◽

Cameron D Kennedy ◽

Vivienne M Velasco ◽

Tory L McGrath ◽

Juliani Weko ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Biological Activity ◽

Cancer Progression ◽

Birth Defects ◽

Mitotic Chromosome ◽

P Element ◽

Inverse Pcr ◽

Gtpase Activating Protein ◽

New Genes ◽

Genomic Analyses

Abstract Faithful chromosome inheritance is a fundamental biological activity and errors contribute to birth defects and cancer progression. We have performed a P-element screen in Drosophila melanogaster with the aim of identifying novel candidate genes involved in inheritance. We used a “sensitized” minichromosome substrate (J21A) to screen ∼3,000 new P-element lines for dominant effects on chromosome inheritance and recovered 78 Sensitized chromosome inheritance modifiers (Scim). Of these, 69 decreased minichromosome inheritance while 9 increased minichromosome inheritance. Fourteen mutations are lethal or semilethal when homozygous and all exhibit dramatic mitotic defects. Inverse PCR combined with genomic analyses identified P insertions within or close to genes with previously described inheritance functions, including wings apart-like (wapl), centrosomin (cnn), and pavarotti (pav). Further, lethal insertions in replication factor complex 4 (rfc4) and GTPase-activating protein 1 (Gap1) exhibit specific mitotic chromosome defects, discovering previously unknown roles for these proteins in chromosome inheritance. The majority of the lines represent mutations in previously uncharacterized loci, many of which have human homologs, and we anticipate that this collection will provide a rich source of mutations in new genes required for chromosome inheritance in metazoans.

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Restriction of P-element insertions at the Notch locus of Drosophila melanogaster

Molecular and Cellular Biology ◽

10.1128/mcb.7.4.1545-1548.1987 ◽

1987 ◽

Vol 7 (4) ◽

pp. 1545-1548

Author(s):

M R Kelley ◽

S Kidd ◽

R L Berg ◽

M W Young

Keyword(s):

Drosophila Melanogaster ◽

Dna Sequences ◽

Consensus Sequence ◽

P Element ◽

Induced Mutations ◽

P Elements ◽

Target Dna ◽

Complex Locus ◽

Additional Level ◽

Derivatives Of

P elements move about the Drosophila melanogaster genome in a nonrandom fashion, preferring some chromosomal targets for insertion over others (J. C. J. Eeken, F. H. Sobels, V. Hyland, and A. P. Schalet, Mutat. Res. 150:261-275, 1985; W. R. Engels, Annu. Rev. Genet. 17:315-344, 1983; M. D. Golubovsky, Y. N. Ivanov, and M. M. Green, Proc. Natl. Acad. Sci. USA 74:2973-2975, 1977; M. J. Simmons and J. K. Lim, Proc. Natl. Acad. Sci. USA 77:6042-6046, 1980). Some of this specificity may be due to recognition of a particular DNA sequence in the target DNA; derivatives of an 8-base-pair consensus sequence are occupied by these transposable elements at many different chromosomal locations (K. O'Hare and G. M. Rubin, Cell 34:25-36, 1983). An additional level of specificity of P-element insertions is described in this paper. Of 14 mutations induced in the complex locus Notch by hybrid dysgenesis, 13 involved P-element insertions at or near the transcription start site of the gene. This clustering was not seen in other transposable element-induced mutations of Notch. DNA sequences homologous to the previously described consensus target for P-element insertion are not preferentially located in this region of the locus. The choice of a chromosomal site for integration appears to be based on more subtle variations in chromosome structure that are probably associated with activation or expression of the target gene.

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PROPERTIES AND EVOLUTIONARY POTENTIAL OF NEWLY INDUCED TANDEM DUPLICATIONS IN DROSOPHILA MELANOGASTER

Genetics ◽

10.1093/genetics/102.1.75 ◽

1982 ◽

Vol 102 (1) ◽

pp. 75-89

Author(s):

Paul A Roberts ◽

David J Broderick

Keyword(s):

Drosophila Melanogaster ◽

Linkage Disequilibrium ◽

Tandem Duplication ◽

Polytene Chromosomes ◽

Primary Source ◽

Evolutionary Potential ◽

X Ray ◽

Fitness Effects ◽

New Genes ◽

Tandem Duplications

ABSTRACT Most of some 33 X-ray-induced duplications recovered as Suppressors of Minute loci proved to be direct tandem duplications. When heterozygous, most duplications were crossover suppressors, and duplications of short to moderate size did not reduce the fitness of their bearers. Crossover suppression by tandem duplication may be attributed to intrastrand foldbacks of the type regularly seen in somatic polytene chromosomes. As a consequence, linkage disequilibrium between duplicated elements and normal chromosomes should be more profound than has been supposed. Tandem duplications appear to be predisposed by reason of frequency of generation, crossover suppression and fitness effects to serve as the primary source of new genes.

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Sequences involved in temperature and ecdysterone-induced transcription are located in separate regions of a Drosophila melanogaster heat shock gene.

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.663 ◽

1986 ◽

Vol 6 (2) ◽

pp. 663-673 ◽

Cited By ~ 32

Author(s):

E Hoffman ◽

V Corces

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Dna Sequences ◽

Germ Line ◽

Initiation Site ◽

P Element ◽

Heat Shock Gene ◽

Base Pairs ◽

Consensus Sequences ◽

Shock Gene

The transcriptional regulation of the Drosophila melanogaster hsp27 (also called hsp28) gene was studied by introducing altered genes into the germ line by P element-mediated transformation. DNA sequences upstream of the gene were defined with respect to their effect on steroid hormone-induced and heat-induced transcription. These two types of control were found to be separable; the sequences responsible for 80% of heat-induced expression were located more than 1.1 kilobases upstream of the RNA initiation site, while the sequences responsible for the majority of ecdysterone induction were positioned downstream of the site at -227 base pairs. We have determined the DNA sequence of the intergenic region separating hsp23 and hsp27 and have located putative heat shock and ecdysterone consensus sequences. Our results indicate that the heat shock promoter of the hsp27 gene is organized quite differently from that of hsp70.

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The Gene Search System: A Method for Efficient Detection and Rapid Molecular Identification of Genes in Drosophila melanogaster

Genetics ◽

10.1093/genetics/151.2.725 ◽

1999 ◽

Vol 151 (2) ◽

pp. 725-737 ◽

Cited By ~ 6

Author(s):

Gakuta Toba ◽

Takashi Ohsako ◽

Naomasa Miyata ◽

Tsuyoshi Ohtsuka ◽

Ki-Hyeon Seong ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Core Promoter ◽

Sequence Similarity ◽

Direct Sequencing ◽

P Element ◽

Drosophila Genome ◽

Coding Region ◽

New Genes ◽

Gene Search ◽

Efficient Detection

Abstract We have constructed a P-element-based gene search vector for efficient detection of genes in Drosophila melanogaster. The vector contains two copies of the upstream activating sequence (UAS) enhancer adjacent to a core promoter, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the basis of phenotypic changes caused by GAL4-dependent forced expression of vector-flanking DNA, and the transcripts were identified with reverse transcriptase PCR (RT-PCR) using the vector-specific primer and followed by direct sequencing. The system had a greater sensitivity than those already in use for gain-of-function screening: 64% of the vector insertion lines (394/613) showed phenotypes with forced expression of vector-flanking DNA, such as lethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% matched the sequences of cloned genes, and 18% matched reported expressed sequence tags (ESTs). Of the insertions in cloned genes, 83% were upstream of the protein-coding region. We discovered two new genes that showed sequence similarity to human genes, Ras-related protein 2 and microsomal glutathione S-transferase. The system can be useful as a tool for the functional mapping of the Drosophila genome.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1520 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1520-1528 ◽

Cited By ~ 14

Author(s):

D Y Chang ◽

B Wisely ◽

S M Huang ◽

R A Voelker

Keyword(s):

Drosophila Melanogaster ◽

Dna Sequences ◽

Insertion Site ◽

Hybrid Dysgenesis ◽

P Element ◽

Dna Transformation ◽

Wild Type ◽

Induced Mutations ◽

X Ray ◽

Element Insertion

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).

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P-element-induced control mutations at the r gene of Drosophila melanogaster.

Molecular and Cellular Biology ◽

10.1128/mcb.5.10.2567 ◽

1985 ◽

Vol 5 (10) ◽

pp. 2567-2574 ◽

Cited By ~ 25

Author(s):

S Tsubota ◽

M Ashburner ◽

P Schedl

Keyword(s):

Drosophila Melanogaster ◽

The Other ◽

P Element ◽

Female Sterility ◽

R Gene ◽

Temporal Expression ◽

Base Pairs ◽

P Elements ◽

Adult Females ◽

Regulatory Mutations

The P-M hybrid dysgenesis system was used to produce five putative regulatory mutations at the rudimentary locus, r. All five mutations were the result of insertions at the 5' end of the gene, upstream of the proposed start of transcription. All of the mutants displayed a leaky wing phenotype, and four of the mutants showed an uncoupling of the wing and female-sterility phenotypes, suggesting that they altered the normal spatial and temporal expression of the r gene. Four of the insertions were P elements. The fifth insertion, which was larger than an intact P element, consisted of a small P element connected to non-P-element DNA. Two of the mutants produced very little r transcript in adult females and were clustered 80 to 150 base pairs upstream of the start of transcription. The other three mutants had higher levels of r transcript in adult females and were clustered 440 to 500 base pairs upstream of the start of transcription. All of the data suggest that the insertions are in a 5' noncoding region of the r gene involved in the control of its spatial and temporal expression.

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The relationship between P elements and male recombination in Drosophila melanogaster.

Genetics ◽

10.1093/genetics/124.2.317 ◽

1990 ◽

Vol 124 (2) ◽

pp. 317-329

Author(s):

A Duttaroy ◽

M McCarron ◽

K Sitaraman ◽

G Doughty ◽

A Chovnick

Keyword(s):

Drosophila Melanogaster ◽

Hot Spot ◽

The Other ◽

P Element ◽

Male Recombination ◽

P Elements ◽

Selective System ◽

Element Mobilization ◽

The Relationship ◽

Initial Binding

Abstract P element dysgenesis associated male recombination in Drosophila was examined with a selective system focused upon 5% of the standard female genetic map divided into eight recombination segments. We found no correspondence between P element mobilization events and recombination in males in the intervals monitored. We defined two adjacent short genetic and molecular regions, one devoid of male recombination and the other acting as a "hot spot" for exchange in the absence of supporting P element insertion and excision activity. These data suggest that, even in the presence of mobilizing P elements, transposase may be active at non-P element sites, and that the genome may harbor sequences ranging from highly responsive to completely unresponsive to transposase action. A viewpoint is presented wherein P elements, with sequences that bind transposase, serve to focus the recombination action of transposase to encompass a region of DNA radiating outward from the initial binding site. We suggest that this region is measured in terms of chromosomal segments rather than limited to P element sequences.

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Genetic Transformation of Drosophila melanogaster with an Autonomous P Element: Phenotypic and Molecular Analyses of Long-Established Transformed Lines

Genetics ◽

10.1093/genetics/115.4.711 ◽

1987 ◽

Vol 115 (4) ◽

pp. 711-723

Author(s):

Stephen B Daniels ◽

Stephen H Clark ◽

Margaret G Kidwell ◽

Arthur Chovnick

Keyword(s):

Drosophila Melanogaster ◽

The Other ◽

P Element ◽

Stable Configuration ◽

Phenotypic Properties ◽

Molecular Analyses ◽

P Elements ◽

Regulatory Ability ◽

Rate Of Increase

ABSTRACT Following transformation of a Drosophila melanogaster true M strain with an autonomous P element, six lines were established and monitored for their molecular and phenotypic properties during a 4-yr period. The number of P elements increased with time in all the lines but the rate of increase differed among lines. Furthermore, degenerate elements arose in each of the lines during propagation. By the end of the 4th yr, the total number of elements in every line was similar to that of a very strong P strain.—At the phenotypic level, all of the transformed lines evolved high P activity, but only three developed complete or nearly complete regulatory ability. The other three lines attained only intermediate levels of regulation over the 4-yr period. One of these lines was particularly noteworthy. Although it contained as many as 55 P elements per genome (20 of which were potentially complete) and had extremely high P activity potential, it continued to exhibit limited regulatory ability. In addition, when females of this line were maintained at high temperatures, the ability to suppress P activity was even further diminished. A strain with this combination of molecular and phenotypic properties, in an apparently stable configuration, has not been previously described.—The results are discussed in the context of the possible role of degenerate elements in regulating P element expression.

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