Human testis expresses a specific poly(A)-binding protein.
(1/1532)
In testis mRNA stability and translation initiation are extensively under the control of poly(A)-binding proteins (PABP). Here we have cloned a new human testis-specific PABP (PABP3) of 631 amino acids (70.1 kDa) with 92.5% identical residues to the ubiquitous PABP1. A northern blot of multiple human tissues hybridised with PABP3- and PABP1-specific oligonucleotide probes revealed two PABP3 mRNAs (2.1 and 2.5 kb) detected only in testis, whereas PABP1 mRNA (3.2 kb) was present in all tested tissues. In human adult testis, PABP3 mRNA expression was restricted to round spermatids, whereas PABP1 was expressed in these cells as well as in pachytene spermatocytes. PABP3-specific antibodies identified a protein of 70 kDa in human testis extracts. This protein binds poly(A) with a slightly lower affinity as compared to PABP1. The human PABP3 gene is intronless with a transcription start site 61 nt upstream from the initiation codon. A sequence of 256 bp upstream from the transcription start site drives the promoter activity of PABP3 and its tissue-specific expression. The expression of PABP3 might be a way to bypass PABP1 translational repression and to produce the amount of PABP needed for active mRNA translation in spermatids. (+info)
Overexpression of eIF4E in Saccharomyces cerevisiae causes slow growth and decreased alpha-factor response through alterations in CLN3 expression.
(2/1532)
The association of G(1) cyclins and Cdc28/cyclin-dependent protein kinase catalyzes the cell cycle entry (Start) in budding yeast. Activation of Start is presumed to be triggered by a post-transcriptional increase in Cln3 during early G(1). Cells arrested by mating pheromone show a loss of cyclin-dependent protein kinase activity caused by transcriptional shutoff of cyclins and/or inhibition by Far1. We report that overexpression of eIF4E (Cdc33), a rate-limiting translation initiation factor, causes an increase in CLN3 mRNA translation, which results in increased expression of CLN2 and in slow growth and decreased alpha-factor response. This phenotype was abrogated in a Deltacln3 or Deltacln2 background. We isolated the transcription factor MBP1 as a multicopy suppressor of the growth and alpha-factor response defects. Furthermore, elevated MBP1, a transcriptional regulator of cyclins, altered the transcriptional start site in CLN3 mRNA, shifting it 45 nucleotides upstream of the normal. This lengthened 5'-untranslated region likely reduces translation efficiency and down-regulates CLN3 protein synthesis, thereby correcting for the excess translation promoted by elevated Cdc33. In addition, the CLN2 mRNA level returned to normal. We propose that regulation of translation initiation by Cdc33 plays a pivotal role in the activation of Start and cell cycle progression in budding yeast. (+info)
Identification of TSIX, encoding an RNA antisense to human XIST, reveals differences from its murine counterpart: implications for X inactivation.
(3/1532)
X inactivation is the mammalian method for X-chromosome dosage compensation, but some features of this developmental process vary among mammals. Such species variations provide insights into the essential components of the pathway. Tsix encodes a transcript antisense to the murine Xist transcript and is expressed in the mouse embryo only during the initial stages of X inactivation; it has been shown to play a role in imprinted X inactivation in the mouse placenta. We have identified its counterpart within the human X inactivation center (XIC). Human TSIX produces a >30-kb transcript that is expressed only in cells of fetal origin; it is expressed from human XIC transgenes in mouse embryonic stem cells and from human embryoid-body-derived cells, but not from human adult somatic cells. Differences in the structure of human and murine genes indicate that human TSIX was truncated during evolution. These differences could explain the fact that X inactivation is not imprinted in human placenta, and they raise questions about the role of TSIX in random X inactivation. (+info)
Structure of the 5' region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites.
(4/1532)
The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5' region of the transcription unit of these genes as well as localization of the 'cis' elements conferring their testis-specific expression gave contradictory results [Widlak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191-200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310-321]. In the present paper we solve these controversies and show that the 5' untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5' UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360 bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on 'cis' regulatory elements localized within exon 1 and the intron sequences. (+info)
Transcriptional regulation of the Bacillus subtilis bscR-CYP102A3 operon by the BscR repressor and differential induction of cytochrome CYP102A3 expression by oleic acid and palmitate.
(5/1532)
The adjacent yrhI and yrhJ genes were identified by the Bacillus subtilis genome sequencing project. We now report that yrhJ (renamed CYP102A3) encodes a cytochrome P450 and that yrhI (renamed bscR) encodes a repressor that negatively regulates the transcription of the bscR-CYP102A3 operon. The transcriptional initiation site of bscR has been mapped by primer extension analysis. An 18-bp perfect palindromic sequence centered 65.5 bp downstream from the transcriptional initiation site of bscR has been identified as the binding site for BscR by gel mobility shift assays. Base substitutions in the 18-bp inverted repeat resulted in derepression of the bscR-xylE transcriptional fusion in vivo. bscR-xylE fusion studies and Northern blot analysis revealed that oleic acid and palmitate could induce the expression of the bscR-CYP102A3 operon to a considerable extent. However, only oleic acid was capable of preventing the binding of BscR to its operator DNA in vitro, suggesting that the induction of CYP102A3 expression by oleic acid and palmitate in B. subtilis might be mediated through different mechanisms. (+info)
Initiator recognition in a primitive eukaryote: IBP39, an initiator-binding protein from Trichomonas vaginalis.
(6/1532)
While considerable progress has been made in understanding the mechanisms of transcription in higher eukaryotes, transcription in single-celled, primitive eukaryotes remains poorly understood. Promoters of protein-encoding genes in the parasitic protist Trichomonas vaginalis, which represents one of the deepest-branching eukaryotic lineages, have a bipartite structure with gene-specific regulatory elements and a conserved core promoter encompassing the transcription start site. Core promoters in T. vaginalis appear to consist solely of a highly conserved initiator (Inr) element that is both a structural and a functional homologue of its metazoan counterpart. Using DNA affinity chromatography, we have isolated an Inr-binding protein from T. vaginalis. Cloning of the gene encoding the Inr binding protein identified a novel 39-kDa protein (IBP39). We show that IBP39 binds to both double and single Inr motifs found in T. vaginalis genes and that binding requires the conserved nucleotides necessary for Inr function in vivo. Analyses of the cloned IBP39 gene revealed no homology at the protein sequence level with identified proteins in other organisms or the presence of known DNA-binding domains. The relationship between IBP39 and Inr-binding proteins in metazoa presents interesting evolutionary questions. (+info)
Characterization of the human and mouse genes for the alpha subunit of type II prolyl 4-hydroxylase. Identification of a previously unknown alternatively spliced exon and its expression in various tissues.
(7/1532)
Prolyl 4-hydroxylase (4-PH) catalyzes the formation of 4-hydroxyproline in -X-Pro-Gly- sequences and has a central role in the synthesis of all collagens. We report here on the cloning and characterization of the genes encoding the catalytic alpha(II) subunits of the human and mouse type II 4-PH [alpha(II)]2beta2 tetramers. The human and mouse genes are approximately 34.6 kb and 30.3 kb in size, respectively, and both consist of 16 exons. The translation initiation codons are located in exon 2, and the sizes of the exons consisting entirely of coding sequences are conserved in the two genes, varying from 54 to 240 bp, whereas the exons 1, containing the transcription initiation sites and 5' untranslated sequences, are 546 bp and 293 bp in the human and mouse, respectively. The sizes of the introns vary from 48 to 49 bp to over 8 kb in both genes. The 5' flanking regions contain no TATA box, but they and introns 1 contain several motifs that may act as transcription factor binding sites, including those for Sox9, which regulates chondrocyte-specific expression of collagens II, IX and XI. Unlike the human alpha(I) gene, the alpha(II) genes do not contain an alternatively spliced exon homologous to exon 9. However, a novel mutually exclusively spliced alternative exon 12a was identified in both genes. The nucleotide and amino-acid sequence identities between the 60-bp exon 12a and 66-bp exon 12b are about 35% and 45%, respectively, in both human and mouse genes. PCR analyses showed that both types of exon 12 are expressed in all tissues studied, except for adult leukocytes that expressed only mRNAs containing exon 12b sequences. Insect cell expression studies showed that a recombinant alpha(II) subunit containing amino acids coded by exon 12a associated with the beta subunit to form a fully active enzyme tetramer. (+info)
Sp1 acts as a repressor of the human adenine nucleotide translocase-2 (ANT2) promoter.
(8/1532)
The human adenine nucleotide translocator-2 promoter is activated by adjacent Sp1 activation elements centered at nucleotides -79 and -68 (Abox and Bbox, respectively), and is repressed by Sp1 bound to a GC element (Cbox) that lies adjacent to transcription start. Here, we address the mechanism of this unique Sp1-mediated repression using transfected Drosophila SL2 and mammalian cell lines. We show that repression is not due to steric interference with assembly of the transcription machinery, as Sp1 bound to the Cbox can, under certain conditions, activate the promoter. Furthermore, ectopic expression of Sp1 deletion mutants in SL2 cells demonstrates that both the Sp1-mediated repression and activation require the D transactivation domain of Sp1 bound to the Cbox. In addition, repression of ABbox-mediated activation is eliminated by separating the Abox and Bbox. Thus, for Cbox-bound Sp1 to repress, Sp1 must be precisely positioned at the region of the ABboxes. Together, these data suggest that the D transactivation domain mediates interactions by Sp1 complexes on separate GC elements that results in repression of the activating Sp1 species. (+info)