Characterization of a cDNA encoding ricin E,a hybrid ricin-Ricinus communis agglutinin gene from the castor plant Ricinus communis

Two classes of ricin cDNA clones have been identified and sequenced. The cDNA clone pBL-1 closely matches in nucleotide sequence the ricin genomic clone pAKG previously described by Halling et al., 1985 (Nucl. Acids Res. 13:8019). A second group of cDNA clones, represented by pBL-3, encode a hybrid protein (ricin E), having a ricin-like A chain and N-terminal half of the B chain and an RCA (Ricinus communis agglutinin)-like C-terminal half of the B chain.     

Characterization of the major mRNAs from adenovirus 2 early region 4 by cDNA cloning and sequencing.

The sequences at the splice junctions of many early region 4 (E4) mRNAs from adenovirus 2 (Ad2) were determined by analysis of cDNA clones. The cDNAs were synthesized from poly(A)+ mRNA isolated from HeLa cells early during Ad2 infection. A standard library was constructed, in pBR322, from double stranded cDNAs initiated by oligo-dT priming. Approximately 1% of total recombinants contained E4 sequences, however among eighty clones analyzed in detail, only four contained the 5' leader sequence. A second library was prepared using a new method that led to a greatly increased representation of desired clones. This method employed oligo-dT to prime the synthesis of the first strand and an oligonucleotide ligated to pBR322, whose sequence was present in the 5' leader, to prime the synthesis of the second strand. With this method the percentage of recombinants containing E4 sequences ranged between 15 and 50% of the total colonies. Virtually all of these E4 cDNA clones contained the 5' leader sequence and several hundred were analyzed by comparing the results from single channel dideoxy sequencing reactions. Nine unique sequence patterns were identified and representative clones were completely sequenced.     

Further analysis of cDNA clones for maize phosphoenolpyruvate carboxylase involved in C4 photosynthesis. Nucleotide sequence of entire open reading frame and evidence for polyadenylation of mRNA at multiple sites in vivo

Four clones of cDNA for phosphoenolpyruvate carboxylase [EC 4.1.1.31] were obtained from a maize green leaf cDNA library by colony hybridization. The largest cDNA was of full-length (3335 nucleotides), being 243 nucleotides longer than the cDNA cloned previously [(1986) Nucleic Acids Res. 14, 1615-1628]. Alignment of the sequence for the N-terminal coding region found in two of the four clones with the sequence reported previously, established the sequence of the entire coding region for the enzyme. The sequencing of 3'-untranslated region of the clones revealed that the poly(A) tract is attached at multiple sites in vivo.     

Isolation and characterization of a partial cDNA clone for heparin cofactor II1

A human fetal liver cDNA library constructed in lambda gt11 was screened with affinity-purified rabbit antibodies raised against heparin cofactor II. One positive clone was plaque purified and the cDNA insert was completely sequenced. The clone encodes the C-terminal 167 amino acid residues of heparin cofactor II as well as the entire 3'-untranslated region of the message. Proline and leucine were identified in the P2 and P1 positions of the protease cleavage site, providing a possible explanation for the ability of heparin cofactor II to inhibit both thrombin and chymotrypsin-like proteases. The coding sequence is identical to that of the recently published human leuserpin 2 (Ragg (1986) Nucl. Acids Res. 14, 1073).     

Structure and expression of the excision repair gene ERCC6, involved in the human disorder Cockayne's syndrome group B.

The human repair gene ERCC6--a presumed DNA (or RNA) helicase--has recently been found to function specifically in preferential nucleotide excision repair (NER). This NER subpathway is primarily directed towards repair of (the transcribed strand of) active genes. Mutations in the ERCC6 gene are responsible for the human hereditary repair disorder Cockayne's syndrome complementation group B, the most common form of the disease. In this report, the genomic organization and expression of this gene are described. It consists of at least 21 exons, together with the promoter covering a region of 82-90 kb on the genome. Postulated functional domains deduced from the predicted amino acid sequence, including 7 distinct helicase signatures, are--with one exception--encoded on separate exons. Consensus splice donor and acceptor sequences are present at all exon borders with the exception of the unusual splice donor at the end of exon VII. The 'invariable' GT dinucleotide in the consensus (C,A)AG/GTPuAGT is replaced by the exceptional GC. Based on 42 GC splice donor sequences identified by an extensive literature search we found a statistically highly significant better 'overall' match of the surrounding nucleotides to the consensus sequence compared to normal GT-sites. This confirms and extends the observation made recently by Jackson (Nucl. Acids Res., 19, 3795-3798 (1991)) derived from analysis of 26 cases. Analysis of ERCC6 cDNA clones revealed the occurrence of alternative polyadenylation, resulting in the (differential) expression of two mRNA molecules (which are barely detectable on Northern blots) of 5 and 7 kb in length.     

Human globin psi B2 is not a globin-related sequence

We have determined the complete nucleotide sequence of 3.4 kilobase pairs of DNA covering the region of the human beta globin gene cluster where a human globin-related sequence psi beta 2 was thought to lie (Fritsch, Lawn, and Maniatis (1980) Cell 19, 959-972). Analysis of the resulting data reveals no evidence for any globin-related sequences in this region. The region does, however, contain several stretches of poly (dA-dT). We have confirmed the observations of Fritsch et al. that DNA from the psi beta 2 region hybridizes to the poly (dA-dT)-tailed human fetal globin cDNA plasmid, pJW151 (Wilson et al., (1978) Nucl. Acids Res. 5, 563-581) under conditions of low stringency, but we find that this hybridization is abolished by the addition of poly(rA). We conclude that psi beta 2 is not a globin pseudogene, and that the earlier investigators were probably misled by hybridization between the poly (dA-dT) stretches within the psi beta 2 region and the tails used in constructing the cDNA plasmid.     

The 19-kDa glycoprotein coded by region E3 of adenovirus. Purification, characterization, and structural analysis

The 19-kDa glycoprotein (gp 19K) coded by early region E3 of adenovirus is of interest as a model for glycoprotein processing and sorting, as well as for the interaction between viral antigens and class I transplantation antigens. In this paper, we show that gp 19K is a major protein synthesized during early stages of infection of human KB cells. We report the purification of gp 19K to near homogeneity, the preparation of a gp 19K antiserum, and structural analyses on the protein. We have determined the DNA sequence of the gp 19K gene in adenovirus type 5 (Ad5) for comparison with the published sequence (Hérissé, J., Courtois, G., and Galibert, F. (1980) Nucleic Acids Res. 8, 2173-2192) of adenovirus type 2 (Ad2). Fragments produced by cyanogen bromide cleavage of Ad2 gp 19K are in accord with the DNA sequence, as are synthetic peptide antibodies targeted to the NH2 terminus of Ad2 gp 19K and the COOH terminus of Ad5 gp 19K. The Ad2 and Ad5 proteins are quite homologous. Conserved features include an NH2-terminal signal sequence, two potential Asn-linked glycosylation sites, and a 20-residue putative transmembrane hydrophobic domain followed by a 15-residue polar domain at the COOH terminus. We show that cleavage of the signal peptide occurs between the 17th and 18th amino acids on both the Ad2 and Ad5 versions of gp 19K and that both potential sites are glycosylated with exclusively high-mannose (as opposed to complex) oligosaccharides. Secondary structure predictions suggest six alpha-helix regions including the signal peptide and transmembrane domain, two or three beta-sheet regions, and about eight beta-turns including the two glycosylation sites and the regions flanking the transmembrane domain.     

mRNAs from human adenovirus 2 early region 4

The molecular structure of the mRNAs from early region 4 of human adenovirus 2 has been studied by Northern blot analysis, S1 nuclease analysis, and sequence analysis of cDNA clones. The results make it possible to identify four different splice donor sites and six different splice acceptor sites. The structure of 12 different mRNAs can be deduced from the analysis. The mRNAs have identical 5' and 3' ends and are thus likely to be processed from a common mRNA precursor by differential splicing. The different mRNA species are formed by the removal of one to three introns, and they all carry a short 5' leader segment. The introns appear to serve two functions; they either place a 5' leader segment in juxtaposition with an open reading frame or fuse two open translational reading frames. The early region 4 mRNAs can encode at least seven unique polypeptides.     

A Kinetic Analysis on the Enhancement of Superoxide Generation from Rice Leaf Disks Stimulated with Proteoglucomannan after Pretreatment with Concanavalin A

We have identified by differential plaque hybridization, human cDNA clones encooding a member of a heat-shock protein family (hsp 90α) in the cDNA library of Adenovirus Type 12 E1A transfected HeLa cells. The complete nucleotide sequence of one of the clones (pHB76–114A) was identified. The sequence of 2912 base pairs had a single reading frame with a coding potential for an 84,672-Da protein. The amino acid sequence was highly homologous, but not identical, to that of the human hsp 90α gene isolated from human peripheral blood lymphocytes [M. Yamazaki, K. Akaogi, T. Miwa, T. Imai, E. Soeda and K. Yokoyama, Nucleic Acids Res., 17, 7108 (1989)]. This cDNA hybridized with RNA species which increased 5- to 20-fold upon heat shock and more than 5-fold in the differentiation stage of human Tera 2 cells.     

Identification of genomic DNA coding for chicken type II procollagen

A segment of the type II procollagen gene has been isolated by screening a lambda Charon 4A library containing fragments of chicken genomic DNA. The specific clone, LgCOL(II), was selected by hybridization using overlapping inserts from two cDNA clones which are specific for a cartilage procollagen (Vuorio, E., Sandell, L., Kravis, D., Sheffield, V. C., Vuorio, T., Dorfman, A., and Upholt, W. B. (1982) Nucleic Acids Res. 10, 1175-1192). DNA sequence analysis of LgCOL(II) in the COOH-telopeptide region of the protein, shows conclusively that this DNA corresponds to the chicken type II procollagen gene. Hybridization of cDNA probes to restriction fragment gel blots together with DNA sequence analysis have established the orientation and position of the procollagen gene within the lambda Charon 4A vector and indicate that LgCOL(II) contains approximately 6 kilobase pairs of the type II procollagen gene plus additional DNA flanking the 3' end of the gene. DNA sequence analysis shows directly that LgCOL(II) contains DNA sequences identical with those in the cDNA clones. The portion of the gene from amino acid 578 of the triple helical region to the COOH-terminal end of the protein (approximately 700 amino acids) is contained within the clone, corresponding to approximately 50% of the amino acid coding sequence of the gene. This region of the chicken alpha 1 (type II) procollagen gene is encoded within a shorter segment of the chicken genome than is the corresponding region of the alpha 2(type I) procollagen gene.     

Sequence of the 3''-noncoding and adjacent coding regions of human gamma-globin mRNA.

In cloning human fetal globin cDNA in bacterial plasmids, we obtained a recombinant which contained a fragment of gammg-globin cDNA corresponding to the region from amino acid 99 to the poly A. We determined a sequence of 169 nucleotides which included the complete 3' non-coding region of the gamma-globin mRNA. The codon for amino acid 136 was GCA, indicating that this cloned fragment was derived from the Agamma-globin gene. In conjunction with the surrounding sequences, the GCA codon provides the Agamma-species with a unique CTGCAG hexanucleotide that is recognized by the restriction enzyme Pst I. The 3'-untranslated region of the gamma-globin mRNA consists of 90 nucleotides, and shares little homology with that of the human beta-globin mRNA. As in other mammalian mRNAs, a symmetrical sequence and the hexanucleotide AAUAAA are present.     

Promoters and processing sites within the transforming region of bovine papillomavirus type 1.

The mRNAs present in bovine papillomavirus type 1 (BPV-1)-transformed C127 cells were studied by primer extension. The results show that two internal promoters are present in the E region of BPV-1 in addition to the previously identified promoter at coordinate 1 (H. Ahola, A. Stenlund, J. Moreno-López, and U. Pettersson, Nucleic Acids Res. 11:2639-2650, 1983). One, located at coordinate 31, generated a set of mRNAs with heterogeneous 5' ends, which may encode the major transforming protein of BPV-1, the E5 protein. The second promoter, which is located at coordinate 39, generates colinear mRNAs which encode either the E4 protein or a truncated form of the E2 protein. Unlike the cottontail rabbit papillomavirus (O. Danos, E. Georges, G. Orth, and M. Yaniv, J. Virol. 53:735-741, 1985), BPV-1 appears to lack a separate promoter for expression of the E7 protein. The major splice sites in the transforming region (E region) of the BPV-1 genome were also identified by nucleotide sequence analysis.     

Human cells express two differentially spliced forms of topoisomerase II beta mRNA.

Screening of a human B-cell cDNA library with a topoisomerase II beta gene-specific probe revealed the presence of two distinct forms of topoisomerase II beta cDNA. One form (designated topoisomerase II beta-1), representing the majority of the clones, would encode the topoisomerase II beta amino acid sequence reported recently [Jenkins, J.R. et al. (1992) Nucleic Acids Res., 20, 5587-5592]. The second form (designated topoisomerase II beta-2) would encode a protein containing an additional 5 amino acids inserted after Valine-23 of the topoisomerase II beta-1 protein sequence. The topoisomerase II beta-1 and beta-2 mRNAs were both widely expressed in human cell lines and tissues. Topoisomerase II beta-2 mRNA was expressed at a lower level than that of the beta-1 form, but the relative expression of the two forms varied in different cell types. Analysis of genomic DNA clones revealed that the two forms of topoisomerase II beta mRNA arose via differential splicing. These data indicate that in addition to the closely related topoisomerase II alpha and beta isozymes, there are two forms of topoisomerase II beta mRNA widely expressed in human cells.     

Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families

A cDNA library has been constructed from an RNA preparation highly enriched in silkmoth chorion mRNAs. Many distinct clones have been identified from this library using a stepwise procedure: scoring for infrequent hexanucleotide restriction enzyme recognition sequences; detailed characterization with restriction enzymes that recognize relatively frequent tetranucleotide sequences; probing the arrangement of the corresponding sequences in chromosomal DNA by the Southern procedure; and detailed cross-hybridization analysis. Unique clones, as well as two classes of distinct but related clones, were revealed by hybridization. The cross-hybridization analysis was greatly facilitated by a newly developed, semiquantitative dot hybridization procedure. The same procedure made it feasible to conveniently estimate the relative abundance of several different sequences in an mRNA mixture. Cloned sequences which scored as relatively abundant in total chorion mRNA were tested with stage-specific chorion mRNA at a very stringent criterion of hybridization. They were thus characterized as early, middle or late sequences with respect to development. The characterized cDNA clones can now be used as probes for studying the evolution, chromosomal organization and regulated developmental expression of the chorion multigene families.     

Molecular cloning of cDNA from foot-and-mouth disease virus C1-Santa Pau (C-S8). Sequence of protein-VP1-coding segment

cDNA segments copied from the RNA of foot-and-mouth disease virus (FMDV) C₁-Santa Pau (isolate C-S8) have been cloned in plasmid pBR322. A 998-bp DNA fragment, that includes the region coding for capsid protein VP1, the carboxy terminus of VP3, and the amino terminus of precursor protein p52 has been sequenced. Comparison of the nucleotide sequence with those from FMDV O₁K, A₁₀61, a₁₂ and C₃ Indaial (Kurz et al., Nucl. Acids Res. 9 (1981) 1919–1931; Kleid et al., Science 214 (1981) 1125–1129; Boothroyd et al., Gene 17 (1982) 153–161; Makoff et al., Nucl. Acids Res. 10 (1982) 8285–8295) indicates extensive variability between the corresponding gene segments, including short insertions and deletions. Base transversions are more frequent than transitions within the VP1 coding segment, but not in the sequence coding for the amino-terminal end of p52. The nucleotide sequence divergence is reflected in variability in both the primary and the predicted higher-order structures of the encoded VP1s.     

Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase

Cytosolic and mitochondrial aspartate aminotransferase cDNAs were cloned from a lambda gt11 rat liver cDNA library. The complete coding sequence and the 3' non-coding sequence of the cytosolic isozyme mRNA were obtained from two overlapping cDNA clones. Partial sequences of the mitochondrial enzyme cDNAs were found to be identical to the recently published complete sequence (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). A single mRNA (2.4 kb (kilobase pair] hybridizing to the mitochondrial cDNA probe was detected by Northern blot analysis, whereas the cytosolic cDNA probe labeled one major (2.1 kb) and two minor (1.8 and 4 kb) mRNAs. The 1.8-kb and the 2.1-kb cytosolic aspartate aminotransferase mRNAs differ in their 3' ends and probably result from the use of either of the two polyadenylation signals present in the 3' noncoding region of the major cytosolic aspartate aminotransferase mRNA. Glucocorticoid hormones increased the activity of cytosolic but not mitochondrial aspartate aminotransferase in both liver and kidney. The increase in the enzyme activity was accompanied by an increase in the amount of the three corresponding mRNAs, while the mitochondrial enzyme mRNA was not significantly modified.