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

We have previously reported concerning the existence of a third type of human alpha-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 alpha-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.     

Human plasma inter-alpha-trypsin inhibitor is encoded by four genes on three chromosomes

Human inter-alpha-trypsin inhibitor is a plasma protein of Mr 180,000 which has long been described as a single polypeptide chain. However, we have previously demonstrated that it is synthesized in liver by two different mRNA populations coding for heavy or light polypeptide chains [Bourguignon, J. et al. (1983) FEBS Lett. 162, 379-383] and cDNA clones for the heavy or light chains have recently been isolated and characterized [Bourguignon, J. et. al. (1985) Biochem. Biophys. Res. Commun. 131, 1146-1153; Salier, J.P. et al. (1987) Proc. Natl Acad. Sci. USA 84, 8272-8276]. In the present study, we show that human poly(A)-rich RNAs hybrid-selected with various heavy-chain-encoding cDNA clones translate three different heavy chains, designated H1 (Mr 92,000), H2 (Mr 98,000) and H3 (Mr 107,000). We previously characterized two heavy-chain cDNA clones. We now report that they correspond to H1 and H2 chains. We have also determined the sequence of an additional cDNA clone which codes for H3 chain. Its insert size is 1.79 kb with a single open reading frame and a poly(A) tail. The deduced amino acid sequence of the H3 chain is highly similar to those of the H1 (54%) and H2 (44%) chains. Northern analysis of human liver poly(A)-rich RNAs with the three heavy-chain cDNAs as probes clearly identified a single major mRNA population of 3.3 +/- 0.1 kb. Chromosomal localization by in situ hybridization shows that inter-alpha-trypsin inhibitor genes are located on three different human chromosomes. The H1 and H3 genes are located in the p211-p212 region of chromosome 3, whereas the H2 gene resides in the p15 band of chromosome 10. The light-chain gene is located in the q32-q33 region of chromosome 9. These results indicate that heavy and light chains of inter-alpha-trypsin inhibitor are encoded by at least four functional genes.     

Murine ferritin heavy chain: isolation and characterization of a functional gene

A mouse liver genomic library screened with a full-length cDNA encoding murine ferritin heavy chain (mFHC) [Torti et al., J. Biol. Chem. 263 (1988) 12638-12644] yielded a functional genomic clone mFHC. The genomic clone isolated included a region of approximately 3 kb containing four exons and three introns. Sequence comparisons of the mouse genomic clone with other genomic clones from rat, human and chicken showed a high degree of similarity among species in the coding regions. Introns and flanking sequences were less conserved. However, comparison of mFHC promoter elements with FHC genes from other species revealed common elements. Analysis of the genomic structure of FHC suggested the presence of pseudogenes. S1 nuclease analysis, however, confirmed that this mouse clone, when transfected into human MRC-5 fibroblasts, was transcribed, indicating that this clone contains an FHC functional gene.     

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.     

Isolation and sequencing of the 5′ end of the rat microtubule-associated protein (MAP1B)-encoding cDNA

We have isolated and sequenced the 5′ end of the cDNA encoding the rat microtubule-associated protein 1B (MAP1B). We found that this region is highly homologous to the corresponding regions of the human [Lien et al., 22 (1994) 273–280] and mouse [Noble et al., J. Cell Biol. 109 (1989) 3367–3376] MAPIB genes. The combination of the sequence that we are presenting with the previously published sequence [Zauner et al., Eur. J. Cell Biol. 57 (1992) 66–74], represents the complete rat MAP1B cDNA coding sequence.     

Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor

U-937 cells, a monocytic line derived from a human histiocytic lymphoma, were induced for human tumor necrosis factor (TNF) secretion into the medium and were used for the preparation of TNF mRNA. Biological activity of the latter was quantified in a Xenopus laevis oocyte injection system. TNF mRNA was enriched by gradient centrifugation and this size-fractionated mRNA was used for synthesis of cDNA and inserted into the unique PstI site of pAT153. A recombinant plasmid containing human TNF cDNA was selected by colony hybridization using an internal fragment of a mouse TNF cDNA clone [Fransen, L., Mueller, R., Marmenout, A., Tavernier, J., Van der Heyden, J., Kawashima, E., Chollet, A., Tizard, R., Van Heuverswyn, H., Van Vliet, A., Ruysschaert, M. R. & Fiers, W. (1985) Nucleic Acids Res. 13, 4417-4429] as a probe. The sequence of this human TNF cDNA is in agreement with the one published by Pennica et al. [Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B. & Goeddel, D. V. (1984) Nature (Lond.) 312, 724-729]. The 157-amino-acid-long mature sequence is about 80% homologous to mouse TNF and its hydrophilicity plot is also very similar, in spite of the apparent species specificity of TNF. In contrast to mouse TNF, it contains no potential N-glycosylation site. When compared to other cytokines, like IFN-beta, IFN-gamma, or IL-2, there is a remarkably high preference for G X C pairs in the third-letter positions. Expression of the TNF cDNA in monkey COS cells or in Escherichia coli gives rise to a protein having similar biological and serological properties as natural human TNF. A human genomic clone was also identified and sequenced; it was found to be in good agreement with the one recently published by Shirai et al. [Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W. & Wallace, R. B. (1985) Nature (Lond.) 313, 803-806], except for some differences in the introns and 5'-untranslated region.     

Molecular cloning and nucleotide sequence of a variant wheat histone H4 gene

To determine whether there is structural variation among histone H4 genes in wheat, one (TH091) of the H4 genes that had been cloned from a wheat genomic DNA library was sequenced and compared with another H4 gene (TH011) which we had described previously [Tabata et al., Nucl. Acids Res. 11 (1983) 5865-5865]. Nucleotide sequence analysis revealed that there are 17 nucleotide replacements in the protein-coding region of two H4 genes, causing only one amino acid substitution: a glycine at position 4 (from the N terminus) in TH011 was replaced by an aspartic acid in TH091. S1 mapping, using total nuclear RNA from germinated seeds, indicated that the H4 gene was transcribed in vivo.     

H3K27 demethylases, at long last

Methylation of lysine 27 on histone H3 (H3K27me) by the Polycomb complex (PRC2) proteins is associated with gene silencing in many developmental processes. A cluster of recent papers (Agger et al., 2007; De Santa et al., 2007; Lan et al., 2007; Lee et al., 2007) identify the JmjC-domain proteins UTX and JMJD3 as H3K27-specific demethylases that remove this methyl mark, enabling the activation of genes involved in animal body patterning and the inflammatory response.     

Bovine dopamine beta-hydroxylase, primary structure determined by cDNA cloning and amino acid sequencing

A cDNA clone encoding bovine dopamine beta-hydroxylase (DBH) has been isolated from bovine adrenal glands. The clone hybridizes to two oligonucleotide probes, one based on a previously reported active site peptide [DeWolf, W. E., Jr., et al. (1988) Biochemistry 27, 9093-9101] and the other based on the human DBH sequence [Lamouroux, A., et al. (1987) EMBO J. 6, 3931-3937]. The clone contains a 1.9-kb open reading frame that codes for the soluble form of bovine DBH, with the exception of the first six amino acids. Direct confirmation of 93% of the cDNA-derived sequence was obtained from cleavage peptides by protein sequencing and mass spectrometry. Differences were found between these two sequences at only two positions. Of the four potential N-linked carbohydrate attachment sites, two, Asn-170 and Asn-552, were shown to be partially and fully glycosylated, respectively. Within the 69% of the protein sequence confirmed by mass spectrometry, no other covalent modifications were detected.     

Linkage of genes for PHI,halothane sensitivity,A-O inhibition,H red blood cell antigens and 6-PGD variants in pigs*

Data from one apparent crossover between S and H, two between PHI and HAL on one side and S on the other, and one between PHI on one side and HAL, S and H on the other, indicate a gene order in pigs of Phi-Hal-S-H-Pgd for genes for PHI, halothane sensitivity, inhibition of expression of A and O, H red blood cell antigens and 6-PGD types. Rasmusen et al. (1980) provided data for a gene order in pigs ofPhi-Hal-H-Pgd for genes for phosphohexose isomerase (PHI) isozyme variants, halothane sensitivity (HAL), H red cell antigens and 6-phosphogluconate dehydrogenase (6-PGD) variants, and suggested that there might be a locus for a gene for inhibition of expression of A and O separate from the locus for H. This is contrary to an earlier proposal by Rasmusen (1972) that the H-system genotype directly influences expression of A and O. Imlah (1980) suggested that the recessive gene for halothane sensitivity has a suppressant effect on the expression of A and O. Andresen (1981) proposed that the locus for inhibition of A and O (for which Rasmusen, 1964, proposed the symbol S) was between the loci for HAL and H types. Data presented in Table 1, which includes haplotypes for three recombinant offspring described by Rasmusen et al. (1980) (883-1, 233-3 and 3864-1) as well as one other recombinant (296-2) provide evidence for the gene order for five genes proposed by Andresen. Types for 6-PGD are listed for all pigs, although they do not provide evidence for gene order in these cases. Male 883-1 (Table 1, and Rasmusen et al., 1980, Table 5) provided the original evidence for recombination between S and H. His phenotype, as well as his genotype as revealed by progeny test (Rasmusen et al., 1980, Table 6) indicated that recombination had occurred between the genes for PHI, HAL and S and the gene for H type in his dam, so that the S locus mapped between H and the loci for the other three traits. The phenotype of one of his sons (233-3, Table 1, and Rasmusen et al., 1980, Table 6) indicated that there had been a recombination between genes for PHI and HAL types on one side and S and H types on the other, providing evidence that the S locus was separate from PHI and HAL as well as H. Another pig listed in Table 1,3864-1, was also described by Rasmusen et al. (1980, Table 9) as a recombinant. This pig provides evidence for recombination between PHI on one side and HAL, S and H on the other, establishing a gene order of Phi-Hal-S-H-Pgd. The last pig listed in Table 1,296-2, is a recombinant comparable to 233-3. The H type of his dam provides markers indicating the recombination was between PHI and HAL on one side and S and H on the other, although the unusual expression of HAL phenotype in both parents of 296-2 makes her haplotypes somewhat uncertain. (Recombination may have been between PHI and HAL rather than as indicated in Table 1.) In spite of incomplete penetrance for HAL (Ollivier et al., 1975; Smith & Bampton, 1977) which makes haplotypes for HAL questionable in some cases, the other genetic markers available are useful to show that recombination has taken place. Without considering the results of halothane testing, if the apparent recombinants are accepted as being as indicated, the order of the genes at the other four loci seems established. Alleles for S types appear to be separable by recombination from those for PHI and H, and the S locus appears to be between the loci for PHI and H. For the five loci, data obtained thus far are cohsistent with a gene order of Phi-Hal-S-H-Pgd.     

Chimeric prion protein expression in cultured cells and transgenic mice.

The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558-1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847-857; Prusiner, S.B., et al., 1990, Cell 63, 673-686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease-resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa-specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.     

Expression of the 53 kD forked protein rescues F-actin bundle formation and mutant bristle phenotypes in Drosophila.

forked mutations affect bristle development in Drosophila pupae, resulting in short, thick, gnarled bristles in the adult. The forked proteins are components of 200-300-microm-long actin fiber bundles that are present transiently during pupal development [Petersen et al., 1994: Genetics 136:173-182]. These bundles are composed of segments of 3-10 microm long, and forked protein is localized along the actin fiber bundle segments and accumulates at the junctions connecting them longitudinally. In the forked mutants, f(36a) and f(hd), F-actin bundles are greatly reduced in number and size, and bundle segmentation is absent. The p-element, P[w(+), falter] contains a 5.3-kb fragment of the forked gene that encodes the 53-kD forked protein [Lankenau et al., 1996: Mol Cell Biol 16:3535-3544]. Expression of only the 53-kD forked protein is sufficient to rescue the actin bundle and bristle phenotypes of f(36a) and f(hd) mutant flies. The 5.3-kb forked sequence, although smaller than the 13-kb region previously shown to rescue forked mutants [Petersen et al., 1994: Genetics 136:173-182], does contain the core forked sequence that encodes actin binding and bundling domains in cultured mammalian cells [Grieshaber and Petersen, 1999: J Cell Sci 112:2203-2211]. These data show that the 53-kD forked protein is sufficient for normal bristle development and that the domains shown previously to be important for actin bundling in cell culture may be all that are required for normal actin bundle formation in developing Drosophila bristles.     

Two base changes restore infectivity to a noninfectious molecular clone of Moloney murine leukemia virus (pMLV-1).

The complete nucleotide sequence of a molecular clone of Moloney murine leukemia virus (pMLV-1) has previously been reported (Shinnick et al., Nature [London] 293:543-548, 1981). However, pMLV-1 does not generate infectious virus after transfection into cells (Berns et al., J. Virol. 36:254-263, 1980). The lesion in pMLV-1 has been localized by determining the biological activity of recombinants containing DNA from an infectious clone of Moloney murine leukemia virus (pMLV-48) and pMLV-1. Replacement of a 1.0-kilobase pair region which spans the gag-pol junction of pMLV-1 with the corresponding DNA fragment from the infectious clone restores its infectivity. Nucleotide sequence analysis of this fragment obtained from the infectious clone (pMLV-48) and pMLV-1 reveals two single base pair changes, one in the p30gag gene and the other in the 5' end of the pol gene. The mutation in the pol gene does not affect the production of infectious virus but renders them XC negative, whereas the mutation in the gag gene appears to be lethal. The complete nucleotide sequence of an infectious clone of Moloney murine leukemia virus can now be deduced.     

Identification of a new polypeptide coded by reovirus gene S1.

The reovirus S1 gene has recently been shown potentially to encode two polypeptides (from two overlapping reading frames) having predicted molecular weights of 49,071 and 16,143 (Nagata et al., Nucleic Acids Res. 12:8699-8710, 1984; Bassel-Duby et al., Nature [London], in press). The larger polypeptide is reovirus protein sigma 1, but synthesis of the smaller polypeptide has not been described to date. A truncated clone of the S1 gene in which the first ATG is deleted was expressed in an in vitro protein synthesis system to yield a approximately 13-kilodalton polypeptide, as determined from migration on sodium dodecyl sulfate-polyacrylamide gels. A polypeptide with a similar migration pattern on sodium dodecyl sulfate-polyacrylamide gels was present in reovirus-infected cells and absent from mock-infected cells. Comparative tryptic peptide analysis of the 13-kilodalton polypeptides produced in vivo and in vitro showed them to be identical. Thus, the s1 mRNA of reovirus type 3 is apparently bicistronic, and we suggest that the approximately 13-kilodalton polypeptide be called sigma s (standing for sigma small).     

Structure and evolution of a mouse tRNA gene cluster encoding tRNAAsp, tRNAGly and tRNAGlu and an unlinked, solitary gene encoding tRNAAsp

We have sequenced mouse tRNA genes from two recombinant lambda phage. An 1800 bp sequence from one phage contains 3 tRNA genes, potentially encoding tRNAAsp, tRNAGly, and tRNAGlu, separated by spacer sequences of 587 bp and 436 bp, respectively. The mouse tRNA gene cluster is homologous to a rat sequence (Sekiya et al., 1981, Nucleic Acids Res. 9, 2239-2250). The mouse and rat tRNAAsp and tRNAGly coding regions are identical. The tRNAGlu coding regions differ at two positions. The flanking sequences contain 3 non-homologous areas: a c. 100 bp insertion in the first mouse spacer, short tandemly repeated sequences in the second spacers and unrelated sequences at the 3' ends of the clusters. In contrast, most of the flanking regions are homologous, consisting of strings of consecutive, identical residues (5-17 bp) separated by single base differences and short insertions/deletions. The latter are often associated with short repeats. The homology of the flanking regions is c. 75%, similar to other murine genes. The second lambda clone contains a solitary mouse tRNAAsp gene. The coding region is identical to that of the clustered tRNAAsp gene. The 5' flanking regions of the two genes contain homologous areas (10-25 bp) separated by unrelated sequences. Overall, the flanking regions of the two mouse tRNAAsp genes are less homologous than those of the mouse and rat clusters.     

Further evidence for the protein coding potential of the mouse mammary tumor virus long terminal repeat: nucleotide sequence of an endogenous proviral long terminal repeat

The 3′ half of an endogenous mouse mammary tumor virus from a C3H mouse was cloned in the Charon 4A vector phage. A comparison of the proviral clone with previously published endogenous mouse mammary tumor virus restriction maps identified it as endogenous unit II (J. Cohen and H. Varmus, Nature [London] 278:418-423, 1979), which is present in all inbred mouse strains derived from the original Bagg albino × DBA cross. The nucleotide sequence of the unit II long terminal redundancy (LTR) was determined and compared with the sequence previously determined for the exogenous C3H virus LTR (Donehower et al., J. Virol. 37:226-238, 1981). Virtually all sequence differences between the two LTRs were base substitutions. The total amount of sequence divergence was 6.6%. The large open reading frame reported previously in the exogenous LTR was preserved in the endogenous LTR. In addition, the pattern of sequence divergence was highly nonrandom with respect to the putative amino acid codons of the two open reading frames. Most of the base substitutions in this region resulted in silent or conservative amino acid codon changes. The nonrandom divergence pattern indicates that selective forces are operating on this segment of DNA and argues that the putative protein is functional in the life cycle of mouse mammary tumor virus. Possible roles for the protein and its mode of expression are discussed.