Nucleotide sequence encoding the precursor of the small subunit of ribulose 1,5-bisphosphate carboxylase from Lemna gibba L.G-3

We have sequenced a cDNA clone, pLgSSU, which encodes the small subunit of ribulose 1,5-bisphosphate carboxylase of Lemna gibba L.G-3 a monocot plant. This clone contains a 832 basepair insert which encodes the entire 120 amino acids of the mature small subunit polypeptide (Mr = 14,127). In addition this clone encodes 53 amino acids of the amino terminal transit peptide of the precursor polypeptide and 242 nucleotides of the 3' non-coding region. Comparison of the nucleotide sequence of pLgSSU with Lemna gibba genomic sequences homologous to the 5' end of the cDNA clone suggests that nucleotides encoding four amino-terminal amino acids of the transit peptide are not included in the cDNA clone. The deduced amino acid sequence of the Lemna gibba mature small subunit polypeptide shows 70-75% homology to the reported sequences of other species. The transit peptide amino acid sequence shows less homology to other species. There is 50% homology to the reported soybean sequence and only 25% homology to the transit sequence of another monocot, wheat.     

Skeletal muscle actin mRNA. Characterization of the 3' untranslated region.

Plasmids p749, p106, and p150 contain cDNA inserts complementary to rat skeletal muscle actin mRNA. Nucleotide sequence analysis indicates the following sequence relationships: p749 specifies codons 171 to 360; p150 specifies codons 357 to 374 together with 120 nucleotides of the 3'-non-translated region; p106 specifies the last actin amino acid codon, the termination codon and the entire 3' non-translated region. Plasmid p749 hybridized with RNA extracted from rat skeletal muscle, cardiac muscle, smooth (stomach) muscle, and from brain. It also hybridizes well with RNA extracted from skeletal muscle and brain of dog and chick. Plasmid p106 hybridized specifically with rat striated muscles (skeletal and cardiac muscle) mRNA but not with mRNA from rat stomach and from rat brain. It also hybridized to RNA extracted from skeletal muscle of rabbit and dog but not from chick. Thermal stability of the hybrids and sensitivity to S1 digestion also indicated substantial divergence between the 3' untranslated end of rat and dog skeletal muscle actins. The investigation shows that the coding regions of actin genes are highly conserved, whereas the 3' non-coding regions diverged considerably during evolution. Probes constructed from the 3' non-coding regions of actin mRNAs can be used to identify the various actin mRNA and actin genes.     

Isolation and characterization of cDNA clones for human skeletal muscle alpha actin.

Two cDNA libraries corresponding to polyA+ RNA from human adult skeletal muscle have been constructed by cloning in the PstI site of pBR322. Skeletal alpha actin cDNA clones have been isolated and characterized. Three of these plasmids have overlapping inserts which together contain the complete 5' non-coding and protein-coding region and part of the 3' untranslated region. Determination of the sequence of the cloned cDNA confirms the complete conservation in human of the amino-acid sequence of skeletal alpha actin compared to the rabbit or rat proteins. The 5' untranslated region, but not the 3' untranslated region, shows good homology with the corresponding one in the rat gene. Analysis of changes at silent sites within the protein-coding region suggests that the divergence of skeletal and cardiac alpha actin took place much earlier than the mammalian radiation. The plasmids described here have been used as probes to detect the homologous gene among the about thirty actin sequences present in the human genome.     

Sequence analysis and comparison of cDNAs of the zein multigene family .

The nucleotide sequence of two zein cDNAs in hybrid plasmids A20 and B49 have been determined. The insert in A20 is 921 bp long including a 5' non-coding region of 60 nucleotides, preceded by what is believed to be an artifactual sequence of 41 nucleotides, and a 3' non-coding region of 87 nucleotides. The B49 insert is 467 bp long and includes approximately one-half the protein coding sequence as well as a 3' non-coding region of 97 nucleotides. These sequences have been compared with the previously published sequence of another zein clone, A30 . A20 and A30 , both encoding 19 000 mol. wt. zeins , have approximately 85% homology at the nucleotide level. The B49 sequence, corresponding to a 22 000 mol. wt. zein, has approximately 65% homology to either A20 or A30 . All three zeins share common features including nearly identical amino acid compositions. In addition, the tandem repeats of 20 amino acids first seen in A30 are also present in A20 and B49 .     

Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment.

A 2112-bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3-kb mRNA from human liver and HL-60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12-bp 5' non-coding region, an open reading frame of 1269 bp and an 831-bp 3' non-coding region with a putative polyadenylation signal 25 bp upstream of a 3' poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N-terminal amino acid residues of human prostatic acid phosphatase and the N-terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.     

烟草花叶病毒蚕豆株系外壳蛋白基因及3‘端非编码区的克隆与序列分析

根据烟草花叶病毒Ｕ１株系序列,人工合成引物,用ＲＴ法合成了ｃＤＮＡ后,通过ＰＣＲ技术扩增并克隆了烟草花叶病毒蚕豆株系的外壳蛋白的基因和３‘端非编码区。ＤＮＡ序列测定结果表明,外壳蛋白基因全长４８０个碱基,编码１５８个氨基酸,３’端非编码区全长２０４个碱基,与ＴＭＶ－Ｕ１株系的同源率为１００％。     

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.     

Cloning and sequence analysis of a cDNA for the alpha-globin mRNA of carp, Cyprinus carpio

A cDNA for alpha-globin mRNA of the carp, Cyprinus carpio, was cloned by the method of Okayama and Berg (Mol. Cell. Biol. 2 (1982) 161-170) and its complete nucleotide sequence was determined. The 5' non-coding region contained 23 nucleotides. Following this region, there was an open reading frame encoded with an alpha-globin polypeptide consisting of 142 amino acids. The 3' non-coding region was 88 nucleotides in length, including two copies of the hexanucleotide AATAAA and a poly(A) site of the GC dinucleotide. There were 16 discrepancies between the reported amino acid sequence of the carp alpha-globin chain and the amino acid sequence predicted from the DNA sequence of the clone. The possible explanations for these differences in amino acid sequence are discussed.     

Nucleotide sequence of complementary DNA and derived amino acid sequence of murine complement protein C3

The nucleotide sequences coding for murine complement component C3 have been determined from a cloned genomic DNA fragment and several overlapping cloned complementary DNA fragments. The amino acid sequence of the protein was deduced. The mature beta and alpha subunits contain 642 and 993 amino acids respectively. Including a 24 amino acid signal peptide and four arginines in the beta-alpha transition region, which are probably not contained in the mature protein, the unglycosylated single chain precursor protein preproC3 would have a molecular mass of 186 484 Da and consist of 1663 amino acid residues. The C3 messenger RNA would be composed of a 56 +/- 2 nucleotide long 5' non-translated region, 4992 nucleotides of coding sequence, and a 3' non-translated region of 39 nucleotides, excluding the poly A tail. The beta chain contains only three cysteine residues, the alpha chain 24, ten of which are clustered in the carboxy terminal stretch of 175 amino acids. Two potential carbohydrate attachment sites are predicted for the alpha chain, none for the beta chain. From a comparison with human C3 cDNA sequence (of which over 80% has been determined) an extensive overall sequence homology was observed. Human and murine preproC3 would be of very similar length and share several noteworthy properties: the same order of the subunits in the precursor, the same basic residue multiplet in the beta-alpha transition region, and a glutamine residue in the thioester region. The equivalent position of the known factor I cleavage sites in human C3 alpha could be located in the murine C3 alpha chain and the size and sequence of the resulting peptide were deduced. A comparison of the amino acid sequences of murine C3 and human alpha 2-macroglobulin is given. Several areas of strong sequence homology are observed, and we conclude that the two genes must have evolved from a common ancestor.     

Expression of the genes coding for the skeletal muscle and cardiac actions in the heart

Several types of evidence indicate that the gene coding for the skeletal muscle actin is expressed in the rat heart: 1) A recombinant plasmid containing an insert with a nucleotide sequence identical to that of the homologous region of skeletal muscle actin gene was isolated from a cDNA library prepared on rat cardiac mRNA template. 2) Using specific probes it was found that the hearts of newborn rats contain a significant amount of skeletal muscle actin mRNA. The quantity of this mRNA in the heart decreases during development. 3) The skeletal muscle actin gene is DNAase I sensitive in nuclei from rat heart tissue. A plasmid containing a cDNA insert homologous to a part of the cardiac actin mRNA was isolated and sequenced. It was found that in spite of the great similarity between the amino acid sequence of the skeletal muscle and cardiac actins, the nucleotide sequences of the two mRNAs are considerably divergent. There is only limited sequence homology between the 3' untranslated regions of the two mRNAs. However, there is an extensive sequence homology between the 3' untranslated regions of the rat and human cardiac mRNAs, suggesting a functional role for this region of the gene or mRNA.     

The silkmoth late chorion locus. I. Variation within two paired multigene families

The 140 X 10(3) base late chorion locus of Bombyx mori contains two 15-member multigene families arranged in tightly linked pairs, which are divergently transcribed (the high-cysteine A (HcA) and the high-cysteine B (HcB) families). Previous DNA hybridization experiments have indicated that all members of these gene families contain a complex pattern of shared sequence variation. The sequence analysis in this paper involving all 15 gene pairs allows a comprehensive examination of the nature of this variation. Average sequence homology between gene pairs is: 95% for the protein-encoding regions; 93% for the common 272 base-pair 5' flanking region; 87% for the introns; and 88% for the 3' untranslated regions. Considering the great degree of sequence homology in the coding regions, an unexpectedly high level of variation is found in the deduced protein sequences. Over 50% of the nucleotide substitutions in the protein-encoding regions lead to amino acid replacements, most of which involve a change in charge or effect the secondary structure of the protein. In addition, significant differences in length between the proteins occur in the carboxyl-terminal arm. In both families, the major portion of this arm is composed of Cys-Gly-Gly and Cys-Gly subrepeats forming a (Cys-Gly-Gly)2-(Cys-Gly)2 major repeat. Differences in the number of complete and partial repeats results in deduced protein sequences that contain arms varying from 32 to 54 amino acid residues for members of the HcA family and 14 to 88 residues for the HcB family. The high level of variation in protein composition indicates a lack of strong selective pressure. We suggest the high level of DNA sequence homology maintained by these genes in the coding as well as in the non-coding regions is the result of sequence exchange between family members.     

Characterization of the bovine herpesvirus 4 major immediate-early transcript.

The major immediate-early (IE) RNA of bovine herpesvirus 4 (BHV-4) has been identified and characterized by analyzing cytoplasmic polyadenylated RNA isolated from Madin-Darby bovine kidney cells infected with BHV-4(DN-599) in the presence of cycloheximide. Hybridization of cDNA to Southern blots of viral DNA, Northern (RNA) blot analysis, and S1 nuclease analyses showed that the major BHV-4 IE RNA is a spliced, 1.7-kb RNA, which is transcribed from right to left on the restriction map of the BHV-4 genome from DNA contained in the 8.3-kb HindIII fragment E. The major IE RNA contains three small exons at its 5' end, spliced to a 1.3-kb 3' exon. This RNA is present in much-reduced amounts when cells are infected in the absence of cycloheximide. However, late in infection, the major IE RNA gene region encodes abundant RNAs which differ in structure from the major IE RNA. Nucleotide sequence analysis of the gene encoding the major IE RNA revealed an open reading frame encoding 284 amino acids. A homology search of amino acid sequence data bases showed that a 141-amino-acid region near the amino terminus of the predicted amino acid sequence is similar to sequences near the amino terminus of herpes simplex virus type 1 IE110. This region of homology includes CXXC pairs, which could be involved in zinc finger structures. The region encoding this putative zinc finger domain is also found in RNAs transcribed from this IE region late in infection, but it is spliced to different sequences than those used in IE RNA. Thus, the major IE region of the BHV-4 genome could encode a family of proteins sharing a zinc finger domain.     

Molecular characterisation of the actin gene of the filarial parasite Wuchereria bancrofti

Wuchereria bancrofti is the major cause of lymphatic filariasis in humans. Although it is responsible for this immensely morbid and debilitating disease, very little is known of the basic molecular biology of this parasite, and there is a vast lack of knowledge on its gene organisation. In this study, the actin gene of W. bancrofti has been characterised by sequencing a clone isolated from a genomic DNA library of this parasite. The 5' flanking region had a potential TATA box and a putative mRNA initiation site. The gene had five exons encoding 376 amino acids, and four introns ranging in size from 109 to 190bp. The 3' flanking region had a potential polyadenylation signal with the sequence ATTAAA which is a common natural variant of the conventional sequence AATAAA. The gene was AT-rich, with a GC content of 37.2%. Southern blot analysis of W. bancrofti genomic DNA indicated that the gene is possibly found as a single copy. The actin amino acid sequence of W. bancrofti showed a high degree of homology to the actin of many organisms of different taxonomic groups, but the highest homology was observed with the free-living nematode Plectus acuminatus. This suggests that P. acuminatus may bear a close evolutionary relationship to W. bancrofti.