Cloning, expression, and function of TFC5, the gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIB.

TFC5, the unique and essential gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been cloned. It encodes a 594-amino acid protein (67,688 Da). Escherichia coli-produced B" has been used to reconstitute entirely recombinant TFIIIB that is fully functional for TFIIIC-directed, as well as TATA box-dependent, DNA binding and transcription. The DNase I footprints of entirely recombinant TFIIIB, composed of B", the 67-kDa Brf, and TATA box-binding protein, and TFIIIB reconstituted with natural B" are indistinguishable. A truncated form of B" lacking 39 N-terminal and 107 C-terminal amino acids is also functional for transcription.

Nodulating strains of Rhizobium loti arise through chromosomal symbiotic gene transfer in the environment.

Rhizobia were isolated from nodules off a stand of Lotus corniculatus established with a single inoculant strain, ICMP3153, 7 years earlier in an area devoid of naturalized Rhizobium loti. The isolates showed diversity in growth rate, Spe I fingerprint of genomic DNA, and hybridization pattern to genomic DNA probes. The 19% of isolates that grew at the same rate as strain ICMP3153 were the only isolates that had the same fingerprint as strain ICMP3153. Sequencing of part of the 16S rRNA gene of several diverse isolates confirmed that they were not derived from the inoculant strain. Nevertheless, all non-ICMP3153 strains gave EcoRI and Spe I hybridization patterns identical to ICMP3153 when hybridized to nodulation gene cosmids. Hybridization of digests generated by the very rare cutting enzyme Swa I revealed that the symbiotic DNA region (at least 105 kb) was chromosomally integrated in the strains. The results suggest that the diverse strains arose by transfer of chromosomal symbiotic genes from ICMP3153 to nonsymbiotic rhizobia in the environment.

Sequence analysis of the cis-regulatory regions of the bithorax complex of Drosophila.

The bithorax complex (BX-C) of Drosophila, one of two complexes that act as master regulators of the body plan of the fly, has now been entirely sequenced and comprises approximately 315,000 bp, only 1.4% of which codes for protein. Analysis of this sequence reveals significantly overrepresented DNA motifs of unknown, as well as known, functions in the non-protein-coding portion of the sequence. The following types of motifs in that portion are analyzed: (i) concatamers of mono-, di-, and trinucleotides; (ii) tightly clustered hexanucleotides (spaced < or = 5 bases apart); (iii) direct and reverse repeats longer than 20 bp; and (iv) a number of motifs known from biochemical studies to play a role in the regulation of the BX-C. The hexanucleotide AGATAC is remarkably overrepresented and is surmised to play a role in chromosome pairing. The positions of sites of highly overrepresented motifs are plotted for those that occur at more than five sites in the sequence, when < 0.5 case is expected. Expected values are based on a third-order Markov chain, which is the optimal order for representing the BXCALL sequence.

SPC3, a synthetic peptide derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120, inhibits HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms.

The third variable region (V3 loop) of gp120, the HIV-1 surface envelope glycoprotein, plays a key role in HIV-1 infection and pathogenesis. Recently, we reported that a synthetic multibranched peptide (SPC3) containing eight V3-loop consensus motifs (GPGRAF) inhibited HIV-1 infection in both CD4+ and CD4- susceptible cells. In the present study, we investigated the mechanisms of action of SPC3 in these cell types--i.e., CD4+ lymphocytes and CD4- epithelial cells expressing galactosylceramide (GalCer), an alternative receptor for HIV-1 gp120. We found that SPC3 was a potent inhibitor of HIV-1 infection in CD4+ lymphocytes when added 1 h after initial exposure of the cells to HIV-1, whereas it had no inhibitory effect when present only before and/or during the incubation with HIV-1. These data suggested that SPC3 did not inhibit the binding of HIV-1 to CD4+ lymphocytes but interfered with a post-binding step necessary for virus entry. In agreement with this hypothesis, SPC3 treatment after HIV-1 exposure dramatically reduced the number of infected cells without altering gp120-CD4 interaction or viral gene expression. In contrast, SPC3 blocked HIV-1 entry into CD4-/GalCer+ human colon epithelial cells when present in competition with HIV-1 but had no effect when added after infection. Accordingly, SPC3 was found to inhibit the binding of gp120 to the GalCer receptor. Thus, the data suggest that SPC3 affects HIV-1 infection by two distinct mechanisms: (i) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4-/GalCer+ cells and (ii) post-binding inhibition of HIV-1 entry into CD4+ lymphocytes.

Primary structure of hepatocyte nuclear factor/forkhead homologue 4 and characterization of gene expression in the developing respiratory and reproductive epithelium.

Members of the winged helix/forkhead family of transcription factors are believed to play a role in cell-specific gene expression. A cDNA encoding a member of this family of proteins, termed hepatocyte nuclear factor/forkhead homologue 4 (HFH-4), has been isolated from rat lung and rat testis cDNA libraries. This cDNA contains an open reading frame of 421 amino acids with a conserved DNA binding domain and several potential transactivating regions. During murine lung development, a single species of HFH-4-specific transcript (2.4 kb long) is first detected precisely at the start of the late pseudoglandular stage (embryonic day 14.5) and, by in situ hybridization, is specifically localized to the proximal pulmonary epithelium. The unique temporal and spatial pattern of HFH-4 gene expression in the developing lung defines this protein as a marker for the initiation of bronchial epithelial cell differentiation and suggests that it may play an important role in cell fate determination during lung development. In addition to expression in the pulmonary epithelium, RNA blot analysis reveals 2.4-kb HFH-4 transcripts in the testis and oviduct. By using mice with genetic defects in spermatogenesis, HFH-4 expression in the testis is found to be associated with the appearance of haploid germ cells and in situ hybridization studies indicate that HFH-4 expression is confined to stages I-VII of spermatogenesis. This pattern of HFH-4 gene expression during the early stages of differentiation of haploid germ cells suggests that HFH-4 may play a role in regulating stage-specific gene expression and cell-fate determination during lung development and in spermatogenesis.

Glycosylation of the c-Myc transactivation domain.

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic posttranslational modification composed of a single monosaccharide, GlcNAc, glycosidically composed of a single monosaccharide, GlcNAc, glycosidically linked to the side-chain hydroxyl of serine or threonine residues. Although O-GlcNAc occurs on a myriad of nuclear and cytoplasmic proteins, only a few have thus far been identified. These O-GlcNAc-bearing proteins are also modified by phosphorylation and form reversible multimeric complexes. Here we present evidence for O-GlcNAc glycosylation of the oncoprotein c-Myc, a helix-loop-helix/leucine zipper phosphoprotein that heterodimerizes with Max and participates in the regulation of gene transcription in normal and neoplastic cells. O-GlcNAc modification of c-Myc is shown by three different methods: (i) demonstration of lectin binding to in vitro translated protein using a protein-protein interaction mobility-shift assay; (ii) glycosidase or glycosyltransferase treatment of in vitro translated protein analyzed by lectin affinity chromatography; and (iii) direct characterization of the sugar moieties on purified recombinant protein overexpressed in either insect cells or Chinese hamster ovary cells. Analyses of serial deletion mutants of c-Myc further suggest that the O-GlcNAc site(s) are located within or near the N-terminal transcription activation/malignant transformation domain, a region where mutations of c-Myc that are frequently found in Burkitt and AIDS-related lymphomas cluster.