The lipoprotein lipase-encoding human gene: sequence from intron-6 to intron-9 and presence in intron-7 of a 40-million-year-old Alu sequence.

The complete nucleotide sequence of the 3877-bp segment spanning the 3′ region of intron-6 to the 5′ region of intron-9 of the human lipoprotein lipase (LPL)-encoding ten-exon gene, LPL, is reported. An Alu repeat present in intron-7 was found by sequence analysis to belong to the 40–55-million-year-old Alu-Se subclass.     

Cloning and characterization of a third type of human α-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human α-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229–235; Tomita et al., Gene 76 (1989) 11–18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human α-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively.Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197–1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5′ region; hence the promoter lies far upstream relative to the other two AMY genes.     

The structure of intron 1 in the chicken growth hormone-encoding gene

An additional PstI fragment of 197 bp was found to be present in intron 1 of the gene encoding chicken growth hormone which we reported previously [Tanaka et al., Gene 112 (1992) 235–239].     

A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides

To analyze human tumors for the presence of mutated ras oncogenes, a procedure was developed based on selective hybridization of mutation-specific oligodeoxynucleotide probes to genomic DNA [Bos et al., Nucl. Acids Res. 12 (1984) 9155–9163]. We have improved this procedure both in sensitivity and speed by including an in vitro amplification step of ras-specific sequences. This amplification step has first been described by Saiki et al. [ Science 230 (1985) 1350–1353] and results in a more than 10⁴-fold increase in the sequence which might contain the mutation. Furthermore, we have improved the selectivity of our hybridizations. As a result, mutated ras oncogenes can now be detected with a dot-blot screening procedure requiring less than 1 μg of tumor DNA.     

Sequence of a canine cDNA clone encoding a Ran/TC4 GTP-binding protein

We report the isolation and characterization of a canine cDNA encoding a 216-amino acid GTP-binding protein of the Ras superfamily. The protein is almost identical to the human TC4 [Drivas et al., Mol. Cell. Biol. 10 (1990) 1793–1798] and Ran [Bischoffand Ponstingi, Proc. Natl. Acad. Sci. USA 88 (1991) 10830–10834; Nature 354 (1991) 80–82] proteins, the latter of which has been found to be involved in cell cycle control. Furthermore, the protein is highly similar to the fission yeast spi1 gene product [Matsumoto and Beach, Cell 66 (1991) 347–360]. The high degree of evolutionary conservation in this protein suggests that it plays a vital role in the eukaryotic cell.     

DNA sequence of Nodulin-45 from Lupinus angustifolius

A partial cDNA clone encoding Lupinus angustifolius Nodulin-45 was isolated by differential hybridisation. A genomic clone was also isolated, from which the DNA sequence was obtained for the 5′ end of the gene (including 1.2 kb of 5′ upstream region). The upstream region includes putative cis-elements, found upstream of other nodulin genes. Southern analysis indicates the presence of several Nodulin-45-like sequences in the lupin genome. The Nodulin-45 protein has a putative N-terminal endoplasmic reticulum-type signal sequence and also contains a large glycine-rich repeat sequence. The cDNA sequence is highly homologous to a Nodulin-45 cDNA sequence from Lupinus luteus (Szczyglowski et al., Plant Sci., 65 (1989) 87–95), although major sequence rearrangements are apparent between the L. luteus and L. angustifolius cDNAs.     

Structure determination and evolution of the chicken cDNA and gene encoding prepropancreatic polypeptide

We have previously demonstrated that the C-terminal regions of the rat and human pancreatic polypeptide (PPP) precursors exhibit a high degree of divergence, whereas the N-terminal regions are highly conserved. This blend of structural conservation and divergence in the precursors appears to be caused by splice junction sliding and translational frameshift in the 3'-region of the PPP gene [Yonekura et al., J. Biol. Chem. 263 (1988) 2990–2997]. In the present study, we determined the nucleotide (nt) sequences of the chicken PPP (cPPP) cDNA and gene, and compared them with those of the mammals. In cPPP, the C-terminal region of the precursor is quite heterologous with respect to the rat (rPPP) and human (hPPP) precursors, and this heterogeneity is accentuated by the large deletion in exon 3 of cPPP. Furthermore, mutational accumulation during evolution caused the structural organization of the 3'-region of cPPP to change; cPPP is terminated in exon 3, whereas rPPP and hPPP are terminated in exon 4. Thus, our previous observation regarding the possibility of ‘mosaic evolution’ [Yamamoto et al., J. Biol. Chem. 261 (1986) 6156–6159] of PPP has been extended and confirmed by this study. Available evidence suggests that ‘mosaic evolution’ is a phenomenon unique to PPP, and not to the genes encoding the other members of the PPP family, neuropeptide-Y and peptide-YY.     

A new member of YER057c family in Trypanosoma cruzi is adjacent to an ABC-transporter

Tcp17 is a Trypanosoma cruzi gene located contiguous to the ABC-transporter tcpgp2. The protein contains 160 amino acid residues with a predicted molecular mass of 16.5 kDa. Western blot analysis using a polyclonal antiserum against recombinant TCP17 revealed that the protein is only expressed in the epimastigote form of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. A sequence comparison of TCP17 showed a remarkable homology with a conserved family of prokaryotic and eukaryotic proteins called YER057c whose function has not yet been characterized. Here, we propose a new signature of this family considering the N-terminal: [IV]–X(4)–[AV]–[AP]–X–[AP]–X(3)–Y–X(9)–[LIVF]–X(2)–[SA]–G–[QS], and the C-terminal: [AT]–R–X(2)–[IVFY]–X–[VC]–X(2)–L–P–X(4)–[LIVM]–E–[IVM]–[DE] motifs. Immunofluorescence and immunoelectron microscopy studies suggest that the protein has a wide distribution in the cell, with a higher concentration in the external side of the plasma membrane, on the Golgi complex and on cytoplasmic vacuoles. Although the physiological function of TCP17 is unknown, its conservation in evolution suggests biological relevance in the parasite.     

Spatial regulation of SpMTA metallothionein gene expression in sea urchin embryos by a regulatory cassette in intron 1

The SpMTA metallothionein (MT) gene of the sea urchin Strongylocentrotus purpuratus is restricted in its expression to the aboral ectoderm in gastrulae and pluteus larvae. The proximal 1.6 kb of the 5′-flanking region together with the 1.12-kb first intron of the SpMTA gene are sufficient for its correct cell-type specific expression in transgenic embryos. This restricted spatial expression is largely eliminated by deletion of an interior 405-bp region in the intron. Within this region is a 295-bp, genomically repetitive, transposon-like segment (Nemer et al., 1993), containing several sequence motifs highly homologous to posited regulatory elements in the promoters of other genes (Thiebaud et al., 1990). The P3A and P5 sites in this apparent regulatory cassette were shown through competition to bind with relatively high affinities the same nuclear factors, bound by their counterpart sites in the CyIIIa actin promoter.     

Mutational analysis of a baculovirus major late promoter

We have used a linker-scan mutation strategy to analyze P_cap99, the proximal promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) gene encoding the major capsid protein. A series of recombinant viruses expressing the cat reporter gene under the control of selected mutants of this promoter was constructed. Only mutations that altered bases within a region extending from 8 bp upstream to 6 bp downstream from a TAAG sequence had a significant effect on expression from the late gene promoter. A synthetic promoter consisting of only these 18 bp (P_capmin) was sufficient to direct late expression. Aside from this small region surrounding the TAAG, no evidence for distinct late activating or repressing sequence elements was obtained. Experiments comparing and combining late and very late gene promoter sequences suggest that late expression is intrinsically determined by the presence and immediate context of a TAAG sequence and that very late expression [as previously shown in Ooi et al. J. Mol. Biol. 210 (1989) 721–736] results from additional modulation of TAAG-dependent expression by downstream promoter elements placed in an appropriate context. A compact combination promoter (95 bp), constructed by fusing P_capmin to linker-modified very late polyhedrin promoter, directs strong expression at late and very late times post-infection.