Replication of heterochromatin and structure of polytene chromosomes.

Heterochromatin is characteristically the last portion of the genome to be replicated. In polytene cells, heterochromatic sequences are underreplicated because S phase ends before replication of heterochromatin is completed. Truncated heterochromatic DNAs have been identified in polytene cells of Drosophila and may be the discontinuous molecules that form between fully replicated euchromatic and underreplicated heterochromatic regions of the chromosome. In this report, we characterize the temporal pattern of heterochromatic DNA truncation during development of polytene cells. Underreplication occurred during the first polytene S phase, yet DNA truncation, which was found within heterochromatic sequences of all four Drosophila chromosomes, did not occur until the second polytene S phase. DNA truncation was correlated with underreplication, since increasing the replication of satellite sequences with the cycE(1672) mutation caused decreased production of truncated DNAs. Finally, truncation of heterochromatic DNAs was neither quantitatively nor qualitatively affected by modifiers of position effect variegation including the Y chromosome, Su(var)205(2), parental origin, or temperature. We propose that heterochromatic satellite sequences present a barrier to DNA replication and that replication forks that transiently stall at such barriers in late S phase of diploid cells are left unresolved in the shortened S phase of polytene cells. DNA truncation then occurs in the second polytene S phase, when new replication forks extend to the position of forks left unresolved in the first polytene S phase.

Histone H2A.Z is widely but nonrandomly distributed in chromosomes of Drosophila melanogaster.

Variant histones that differ in amino acid sequence from S-phase histones are widespread in eukaryotes, yet the structural changes they cause to nucleosomes and how those changes affect relevant cellular processes have not been determined. H2A.F/Z is a highly conserved family of H2A variants. H2Av, the H2A.F/Z variant of Drosophila melanogaster, was localized in polytene chromosomes by indirect immunofluorescence and in diploid chromosomes by chromatin immunoprecipitation. H2Av was widely distributed in the genome and not limited to sites of active transcription. H2Av was present in thousands of euchromatic bands and the heterochromatic chromocenter of polytene chromosomes, and the H2Av antibody precipitated both transcribed and nontranscribed genes as well as noncoding euchromatic and heterochromatic sequences. The distribution of H2Av was not uniform. The complex banding pattern of H2Av in polytene chromosomes did not parallel the concentration of DNA, as did the pattern of immunofluorescence using H2A antibodies, and the density of H2Av measured by immunoprecipitation varied between different sequences. Of the sequences assayed, H2Av was least abundant on 1. 688 satellite sequences and most abundant on the hsp70 genes. Finally, transcription caused, to an equivalent extent, both H2Av and H2A to be less tightly associated with DNA.

Characterization of CCK-B/gastrin-like receptors in human gastric carcinoma.

In this study we identified and characterized the receptor related to the modulation of growth of human gastric cancer by gastrin. By performing receptor binding assays on human AGS gastric cancer cells with the selective CCK-B/gastrin receptor antagonist [3H]L-365,260, specific and saturable binding were determined. Binding was dependent on protein concentration, time, temperature, and the presence of protease inhibitors, and was located in the membrane fraction. Gastrin, as well as CCK, stimulated gastric cancer cell growth in a receptor-mediated fashion at a concentration consistent with the binding affinity. Receptor gene expression for the CCK-B/gastrin receptor, but not for the CCK-A receptor, was found by reverse transcription polymerase chain reaction. Receptor binding assays as well as transcriptional and growth studies provide evidence that gastrin-stimulated growth of human gastric cancer is mediated by CCK-B/gastrin-like receptors.

Upstream stimulatory factor family binds to the herpes simplex virus type 1 latency-associated transcript promoter.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) promoter 1 (LP1) is the only viral promoter that exhibits detectable transcriptional activity during a latent HSV infection. The LAT promoter-binding factor (LPBF) regulatory sequence (nucleotides -65 to -72 relative to the transcriptional start site of the 8.3-kb primary transcript) closely resembles the core recognition sequence required for binding members of the upstream stimulatory factor (USF)/major late transcription factor (MLTF) family. In this analysis, we demonstrate that oligonucleotides containing either the LPBF recognition sequence or the USF/MLTF recognition sequences from previously described promoters bind cellular factors which exhibit very similar mobilities in electrophoretic mobility shift (EMS) analyses. We also observe a high degree of similarity in competition profiles obtained in competition EMS analyses utilizing oligonucleotides containing recognition sequences for either LPBF or USF/MLTF. Furthermore, antibody supershift EMS analyses have demonstrated that the factors binding the LPBF or USF/MLTF recognition sites in these oligonucleotides are antigenically related, if not identical, and that greater than 90% of the LPBF-binding activity is antigenically related to USF. In addition, we demonstrate that both forms of in vitro translated USF proteins (43 and 44 kDa) bind to the LPBF recognition sequence within HSV-1 LP1. Taken together, these data indicate that USF is capable of binding to the HSV-1 LPBF recognition sequence and that USF is a major LPBF-binding activity in cells of neuronal and nonneuronal lineage. These data further support the hypothesis that USF may indeed play a significant role in the transcriptional activity of HSV-1 LP1.

Characterization of the CCK-B/gastrin-like receptor in human colon cancer.

The gastrointestinal peptide, gastrin, tonically stimulates growth of human colon cancer cells in vivo and in vitro, and does so in a receptor-mediated fashion. This study defined the nature of gastrin binding in human colon cancer using [3H]L-365,260, a specific cholecystokinin B (CCK-B)/gastrin antagonist found to block gastrin's effects on growth. Following elucidation of optimal binding conditions (e.g., pH, time, and temperature) in log phase HT-29 human colon cancer cells, specific and saturable binding with a dissociation constant of 4.8 +/- 0.7 nM and a maximal binding capacity (Bmax) of 320 +/- 120 fmol/mg protein, consistent with a single binding site, was recorded. Binding was localized to the membrane fraction. Exposure to gastrin or receptor antagonist decreased and increased, respectively, the Bmax. Competition experiments indicated that L-365,260 was 25- and 200-fold more effective at displacing radiolabeled L-365,260 than gastrin and cholecystokinin, respectively. In contrast to log phase cells, the Bmax was decreased by 67 to 76% in confluent and postconfluent cultures. Binding activity was observed in other cell lines examined, as well as in xenografts and colon cancers obtained at surgery. Binding in normal human colonic mucosa was 10-fold less than in colon cancer. These results provide the first comprehensive identification and characterization of a CCK-B/gastrin-like receptor in human colon cancer.