Redistribution of transcription factor AP-2alpha in differentiating cultured human epidermal cells.

Expression of the transcription factor AP-2alpha was examined in cultured human epidermal cells. Levels of AP-2alpha mRNA increased substantially after the cultures reached confluence, similar to the expression pattern of the differentiation markers involucrin and keratinocyte transglutaminase. The level of AP-2alpha protein in nuclear extracts declined markedly after confluence, however, along with its ability to form complexes with oligonucleotides containing the AP-2 response element. In contrast, the levels of AP-2alpha protein in cytoplasmic extracts increased dramatically after confluence, but these extracts had low DNA binding activity. Supershift experiments with specific antisera detected only AP-2alpha and not the beta or gamma isoforms. Examination of its localization by confocal microscopy revealed that AP-2alpha was primarily in the nucleus of basal cells and largely cytoplasmic in the most superficial cells. Localization was a dynamic phenomenon in that changing the medium resulted in accumulation of this transcription factor in the nucleus after several hours. Overall, the data indicate that AP-2alpha transcriptional activity is regulated in a differentiation-dependent manner in cultured keratinocytes and that this occurs by relocalization of the protein. Nuclear localization of the AP-2alpha protein in basal cells permits its accessibility to response elements in gene promoters, whereas sequestration in the cytoplasm as the differentiation program progresses curtails its transcriptional activity. This regulatory scheme may provide keratinocytes with the ability to restore AP-2 transcriptional activity rapidly by redistribution to the nucleus after receiving an appropriate growth signal, such as a medium change.

Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes.

Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.

Dimerization of plasmid DNA accelerates selection for antibiotic resistance.

Dimerization of multicopy plasmids is widely assumed to be disadvantageous both for plasmid maintenance and for the host cell. It is known that dimerization causes plasmid instability; dimer-containing cells grow slower than their monomer-containing counterparts. However, as we demonstrate here, under conditions of selective stress, dimers provide an advantage for bacteria. Dimers facilitate segregation of mutants from numerous copies of the parental plasmid. Accelerated segregation greatly increases the rate of accumulation of plasmids carrying mutations that are adaptive for bacteria. In contrast, resolution of dimers by site-specific recombination decreases, 10(3)-10(5)-fold, the efficiency of selection of spontaneous reversions in the tet gene of pBR327.

[Nature of female sterility in mutants at the ecs locus controlling sensitivity to ecdysterone in Drosophila melanogaster]. / Issledovanie prirody steril'nosti samok u mutantov po lokusu ecs, kontroliruiushchemu chuvstvitel'nost' k ekdisteronu u Drosophila melanogaster.

Using interstrain level ovary transplantations function of ovaries was estimated in females bearing mutations at the ecs locus mapped in the 2B3-5 region of the X-chromosome of Drosophila melanogaster. Dissection of recipient flies and genetic analysis of their progeny demonstrated that the donor ovary in fertile flies was capable of normal functioning in the case of connection of the donor ovary in fertile flies with the recipient oviducts. Therefore, female-sterile mutations at the ecs locus are somatic line specific. SDS-PAGE electrophoresis revealed normal level of the yolk proteins in such females. Thus, the sterility of the ecs mutants may arise from abnormal morphogenesis of their genital disc, due to the loss of normal sensitivity to ecdysterone.

Cytogenetical analysis of the 2B3-4-2B11 region of the X chromosome of Drosophila melanogaster. VII. Influence of the ecs locus on female fertility.

Of 13 ecs mutations, which affect female fertility, as revealed by complementation analysis, 7 are chromosome rearrangements involving the br complementation group. The other six show no cytologically detectable rearrangements and behave as completely or partially noncomplementing ecs alleles. All viable combinations of these 13 mutations were characterized by partial or complete female sterility. Viable heterozygotes carrying any of these mutations and the rearrangements Df(1)sta, T(1;3)sta, Df(1)St490, previously localized distal to the ecs locus, were also sterile. Using deletions and an electrophoretic mobility variant from the Staket strain, a minor chorion gene S70 has been mapped. It had been thought this gene was located in the 2B3-5 region, and corresponded to the ecs locus. However, in the present study, this gene was shown to map in the region removed by Df(1)sta (1E1-2-2B3-4) but outside that removed by Df(1)At127 (1E1-2-2A1-2), i.e. within the 2A1-2-2B3-4 region which is distal to the ecs locus. Rearrangements and point mutations at the ecs locus that result in female sterility had no effect on synthesis of the chorion protein s70. It may therefore be suggested that the chorion protein gene is not functionally associated with the ecs locus and that sterility is caused not by disruptions of the chorion protein gene but by lesions in the ecs gene itself. Thus, an ecs product, which controls cell sensitivity to ecdysterone is also necessary for female fertility.(ABSTRACT TRUNCATED AT 250 WORDS)

[Relationship between the female fertility function of the ecs locus and the gene for a minor chorion s70 protein of Drosophila melanogaster]. / O sviazi funktsii fertil'nosti samok lokusa ecs s genom minornogo khorionovogo belka s70 Drosophila melanogaster.

The staket strain carrying an electrophoretic variant of the minor chorion protein was used to determine the chromosomal location of the s70 gene. The gene was shown to locate within the Df(1)sta (1E1-2-2B3-4) and outside the Df(1)At127 (1E1-2-2A1-2). Therefore, the s70 chorion gene resides within the region 2A1-2-2B3-4 on the X-chromosome, i.e. outside the ecs locus. Female-sterile mutations of the ecs locus do not interfere with expression of the chorion gene.

[The effect of locus ecs in Drosophila melanogaster on fertility of females]. / Vliianie lokusa ecs Drosophila melanogaster na fertil'nost' samok.

A total of 13 ecs mutations affecting female fertility were isolated by complementation analysis. Seven of them were rearrangements with the br complementation group phenotype. Six other mutations had no cytologically detectable rearrangements and behaved as completely or partially non-complementing alleles of the ecs locus. All viable combinations of the above 13 mutations disturbed female fertility. Sterile were all fully viable compounds carrying any of these mutations and rearrangements Df (1) sta, T(1; 3)sta, Df(1)St490, previously localized on the molecular map distally to the ecs locus. According to data on location on molecular map of lesions affecting fertility, at least two elements at the ecs locus seem essential for this function: the most distal (left) cis-acting zone with no effect on viability and a sequence within the limits of the essential part of the ecs locus. Disturbance of any of these zones or their separation in the rearranged chromosomes lead to female sterility.