Spermatid-specific overexpression of the TATA-binding protein gene involves recruitment of two potent testis-specific promoters

The gene encoding the TATA-binding protein, TBP, is highly overexpressed during the haploid stages of spermatogenesis in rodents. RNase protection analyses for mRNAs containing the previously identified first, second, and eighth exons suggested that most TBP mRNAs in testis did not initiate at the first exon used in somatic cells (here designated exon 1C). Using a sensitive ligation-mediated cDNA amplification method, 5' end variants of TBP mRNA were identified, and the corresponding cDNAs were cloned from liver and testis. In liver, a single promoter/first exon is used to generate a steady-state level of roughly five molecules of TBP mRNA per diploid cell equivalent. In testis, we detect modest up-regulation of the somatic promoter and recruitment of at least five other promoters. Three of the alternative promoter/first exons, including 1C and two of the testis-specific promoter/first exons, 1D and 1E, contribute roughly equivalent amounts of mRNA which, in sum, account for greater than 90% of all TBP mRNA in testis. As a result, round spermatids contain an estimated 1000 TBP mRNA molecules per haploid cell. Testis TBP mRNA also exhibits several low abundance 5' end splicing variants; however, all detected TBP mRNA leader sequences splice onto the common exon 2 and are expected to initiate translation at the same site within exon 2. The precise locations of the three major initiation exons are mapped on the gene. The identification of the strong testis-specific promoter/first exons will be important for understanding spermatid-specific tbp gene regulation.     

Differential splicing of the growth hormone-releasing hormone gene in rat placenta generates a novel pre-proGHRH mRNA that encodes a different C-terminal flanking peptide

We have isolated and characterized a novel rat placental pre-proGHRH mRNA (pre-proGHRH-2 mRNA). This mRNA is generated by an alternative splicing process which results in the presence of an additional exon of 156 bp (designated exon 4.5) located between exons 4 and 5 of the previously reported hypothalamic and placental pre-proGHRH mRNA (pre-proGHRH-1 mRNA). Since the sequences encoding mature GHRH are included within exons 3 and 4, the processing of pre-proGHRH-2 would not affect the synthesis of mature GHRH but would generate a C-terminal peptide (designated GCTP-2) different from that previously reported in the hypothalamus and placenta (GCTP-1). The putative GCTP-2 has 64 amino acids, and the first 18 N-terminal residues are identical to those present in GCTP-1 (30 amino acids long). Pre-proGHRH-2 mRNA has not been detected in the hypothalamus.     

Expression of insulin-like growth factor-II and IGF-binding protein-1 mRNAs in term rhesus monkey placenta: comparison with human placenta

The patterns of expression insulin-like growth factor-II (IGF-II) and IGF-binding protein-1 (IGFBP-1) mRNAs were compared between term human and rhesus monkey placenta using in situ hybridization histochemistry. Since IGFs and IGFBPs are paracrine factors, the identification of the sites of synthesis of the IGFs and their binding proteins indicate the potential sites of biological action. In both species, IGF-II mRNA was found in highest abundance in the extravillous cytotrophoblasts. The major difference was observed in placental villi. In the human placenta, IGF-II mRNA was expressed in the chorionic mesoderm of the placental villi, whereas, in the rhesus placenta, it was expressed in the syncytiotrophoblasts and not in the chorionic mesoderm. In both species, IGFBP-1 mRNA was expressed only in the decidua. Therefore, the pattern of expression of IGFBP-1 mRNA in the maternal decidua is similar between rhesus monkey and human placenta, but that of IGF-II mRNA in the fetal placental villi is different. These data suggest that the IGF-II-IGFBP-1 interaction in the paracrine regulation of placental growth and/or function in the rhesus monkey and human placentae may have similarities and differences.     

Imprecise excision of the Caenorhabditis elegans transposon Tc1 creates functional 5' splice sites

Imprecise excision of the Caenorhabditis elegans transposon Tc1 from a specific site of insertion within the unc-54 myosin heavy chain gene generates either wild-type or partial phenotypic revertants. Wild-type revertants and one class of partial revertants contain insertions of four nucleotides in the unc-54 third exon (Tc1 "footprints"). Such revertants express large amounts of functional unc-54 myosin despite having what would appear to be frameshifting insertions in the unc-54 third exon. We demonstrate that these Tc1 footprints act as efficient 5' splice sites for removal of the unc-54 third intron. Splicing of these new 5' splice sites to the normal third intron splice acceptor removes the Tc1 footprint from the mature mRNA and restores the normal translational reading frame. Partial revertant unc-54(r661), which contains a single nucleotide substitution relative to the wild-type gene, is spliced similarly, except that the use of its new 5' splice site creates a frameshift in the mature mRNA rather than removing one. In all of these revertants, two alternative 5' splice sites are available to remove intron 3. We determined the relative efficiency with which each alternative 5' splice site is used by stabilizing frameshifted mRNAs with smg(-) genetic backgrounds. In all cases, the upstream member of the two alternative sites is used preferentially (> 75% utilization). This may reflect an inherent preference of the splicing machinery for the upstream member of two closely spaced 5' splice sites. Creation of new 5' splice sites may be a general characteristic of Tc1 insertion and excision events.