DNA polymerase II is encoded by the DNA damage-inducible dinA gene of Escherichia coli.

The structural gene for DNA polymerase II was cloned by using a synthetic inosine-containing oligonucleotide probe corresponding to 11 amino acids, which were determined by sequencing the amino terminus of the purified protein. The labeled oligonucleotide hybridized specifically to the lambda clone 7H9 from the Kohara collection as well as to plasmid pGW511 containing the SOS-regulated dinA gene. Approximately 1400 base pairs of dinA sequence were determined. The predicted amino-terminal sequence of dinA demonstrated that this gene encoded DNA polymerase II. Sequence analysis of the upstream region localized a LexA binding site overlapping the -35 region of the dinA promoter, and this promoter element was found to be only two nucleotides downstream from the 3' end of the araD gene. These results demonstrate that the gene order is thr-dinA (pol II)-ara-leu on the Escherichia coli chromosome and that the DNA polymerase II structural gene is transcribed in the same direction as the araBAD operon. Based on the analysis of the predicted protein, we have identified a sequence motif Asp-Xaa-Xaa-Ser-Leu-Tyr-Pro-Ser in DNA polymerase II that is highly conserved among a diverse group of DNA polymerases, which include those from humans, yeast, Herpes and vaccinia viruses, and phages T4 and PRD1. The demonstration that DNA polymerase II is a component of the SOS response in E. coli suggests that it plays an important role in DNA repair and/or mutagenesis.     

Vesicular stomatitis virus glycoprotein is anchored in the viral membrane by a hydrophobic domain near the COOH terminus

We have determined the COOH-terminal and NH(2)-terminal amino acid sequences of the vesicular stomatitis virus (VSV) glycoprotein (G). A sequence of 122 COOH-terminal amino acids was deduced from the complete sequence of a cloned DNA insert carrying 470 nucleotides derived from the 3' end of the G mRNA. Evidence presented indicates that this portion of the polypeptide includes the domains of G that reside inside the virion and span the lipid bilayer of the virion. This seems clear because a partial amino acid sequence of a fragment of G that remains associated with the membrane of the virion after exhaustive proteolytic digestions can be located unambiguously in the predicted sequence. This predicted sequence contains an uninterrupted hydrophobic domain beginning 49 amino acids and ending 30 amino acids from the COOH terminus. This region presumably spans the lipid bilayer. The COOH-terminal portion of 29 amino acids contains a high proportion of basic residues and resides inside the virion. The COOH-terminal portion of the VSV G protein therefore resembles in structure that of glycophorin, an erythrocyte membrane protein well characterized previously. The configuration of G in the viral membrane demonstrated here is probably similar for other viral glycoproteins, although this has not been tested as directly in any other case. From the sequence of a DNA primer extended on the RNA genome from the adjacent M protein gene into the G protein gene, we have deduced an NH(2)-terminal G protein sequence of 53 amino acids, including the leader sequence of 16 amino acids. Our sequence confirms, extends, and corrects two partial amino acid sequences reported for this region previously.     

Molecular structure of the human cytoplasmic beta-actin gene: interspecies homology of sequences in the introns.

A recombinant phage that carries the cytoplasmic beta-actin gene was isolated from a human DNA library. The nucleotide sequence of this gene was determined. The amino acid sequence deduced from the nucleotide sequence matches perfectly that of beta-actin from human fibroblasts. The gene contains five introns. A large intron was found in the 5' untranslated region six nucleotides upstream from the ATG initiation codon. Four introns were found within the coding region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. In contrast to the human cardiac muscle actin gene, the aorta-type smooth muscle actin gene, and the stomach-type smooth muscle actin gene, the beta-actin gene lacks the codon for cysteine between the ATG initiation codon and the codon for the NH2-terminal amino acid of the mature protein. Hybridization of genomic DNA with DNA fragments derived from intron I in the 5' untranslated region and from intron III strongly suggests the presence of a single beta-actin gene in the human genome. The DNA sequences of the coding region, of the 3' untranslated region, and of the sequence block between the "CCAAT" box and "TATA" box in the 5' flanking DNA of the human beta-actin gene are highly homologous to the corresponding sequences of the rat and chicken beta-actin genes. Unexpectedly, the sequence of intron III of the human beta-actin gene shows considerable homology to that of the rat beta-actin gene.     

The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence.

The primary structure of the beta chain of human glycoprotein Ib (GPIb), the platelet receptor for von Willebrand factor, has been established by a combination of cDNA cloning and amino acid sequence analysis. A lambda phage cDNA expression library prepared from human erythroleukemia cells (HEL cells) was screened with a radiolabeled affinity-purified rabbit polyclonal antibody to the beta chain of GPIb. Eighteen positive clones were isolated and plaque-purified and the nucleotide sequences of three were determined. The composite sequence spanned 968 nucleotides and included a 5' untranslated region of 22 nucleotides, an open reading frame of 618 nucleotides encoding a signal peptide of 28 amino acids and a mature protein of 181 amino acids, a stop codon, and a 3' noncoding region of 307 nucleotides. The 3' noncoding sequence also contained a polyadenylylation signal (AATAAA) 14 nucleotides upstream from the poly(A) tail of 18 nucleotides. Edman degradation of the intact beta chain and of peptides produced by chemical cleavage yielded amino acid sequences spanning 76 residues that were identical to those predicted from the cDNA. The amino-terminal region of the beta chain contains a leucine-rich sequence of 24 amino acids that is similar to a sequence that occurs as seven tandem repeats in the alpha chain of GPIb and nine tandem repeats in leucine-rich alpha 2-glycoprotein. The leucine-rich sequence in the beta chain of GPIb is flanked on both sides by amino acid sequences that are similar to those flanking the leucine-rich tandem repeats of the alpha chain of GPIb and leucine-rich alpha 2-glycoprotein. The amino-terminal region of the beta chain of GPIb is followed by a transmembrane segment of 25 amino acids and an intracellular segment of 34 amino acids at the carboxyl terminus of the protein. The intracellular segment contains an unpaired cysteine and two potential sites for phosphorylation by cAMP-dependent protein kinase.     

Cloning of a cDNA coding for human factor V, a blood coagulation factor homologous to factor VIII and ceruloplasmin.

Coagulation factor V is a high molecular weight plasma glycoprotein that participates as a cofactor in the conversion of prothrombin to thrombin by factor Xa. A phage lambda gt11 Hep G2 cell cDNA expression library was screened by using an affinity-purified antibody to human factor V, and 11 positive clones were isolated and plaque-purified. The clone containing the largest cDNA insert contained 2970 nucleotides and coded for 938 amino acids, a stop codon, and 155 nucleotides of 3' noncoding sequence including a poly(A) tail. The coding region includes 651 amino acids from the carboxyl terminus that constitute the light chain of human factor Va and 287 amino acids that are part of the connecting region of the protein. The predicted amino acid sequence agreed completely with 147 amino acid residues that were identified by Edman degradation of cyanogen bromide peptides isolated from the light chain. During the activation of factor V, several peptide bonds are cleaved by thrombin, giving rise to a heavy chain, a connecting fragment(s), and a light chain. The light chain is generated by the cleavage of an Arg-Ser peptide bond. The amino acid sequence of the light chain is homologous (40%) with the carboxyl-terminal fragment (Mr, 73,000) of human factor VIII. Both fragments have a similar domain structure that includes a single ceruloplasmin-related domain followed by two C domains. The carboxyl terminus of the connecting region, however, shows no significant amino acid sequence homology with factor VIII. It is very acidic and contains a number of potential N-linked glycosylation sites. It also contains about 20 tandem repeats of nine amino acids.     

Isolation and analysis of genomic DNA clones encoding the third component of mouse complement.

A gene library was constructed with DNA from strain A mice by using the phage lambda vector lambda 1059. By screening with cloned cDNA for the third component of mouse complement, C3, four different C3 genomic clones were isolated from this library. Two of the recombinant phages carry insertions of 14 and 18 kilobase pairs, respectively, which together cover one complete copy of the C3 gene and several hundred nucleotides of its 5' and 3' flanking sequences. The distance from the 5' end of the gene, which includes the hexanucleotide T-A-T-A-A-A and a translation initiation codon, to its 3' end as defined by the poly(A) attachment site is 24 kilobase pairs. From the genomic DNA sequence, a signal peptide of 24 amino acid residues is predicted at the NH2 terminus of the initial translation product. The signal peptide and the next two amino acids are encoded by the first exon of this gene.     

Nucleotide sequence of the Escherichia coli prolipoprotein signal peptidase (lsp) gene.

The nucleotide sequence of the prolipoprotein signal peptidase (lsp) gene has been determined. The lsp gene was found to be adjacent to the isoleucyl-tRNA synthetase ( ileS ) gene, such that the termination codon of the ileS gene overlaps with the initiation codon of lsp. These two genes are transcribed in the same direction and the major promotor for the lsp gene appears to be upstream of ileS . Identification of the lsp gene was established by amplification of prolipoprotein signal peptidase activity in strains carrying a subcloned 1.1-kilobase Stu I-Acc I fragment and was further confirmed by introducing mutational alterations in the COOH terminus of the protein that caused a decrease in prolipoprotein signal peptidase activity. The deduced amino acid sequence indicates that prolipoprotein signal peptidase contains 164 residues. Unlike most exported proteins, there is no apparent signal peptide sequence for the lsp protein. Computer-assisted secondary structure analysis of the deduced amino acid sequence identified four hydrophobic regions that share features common to transmembrane segments in integral membrane proteins.     

Nucleotide sequence and the encoded amino acids of human serum albumin mRNA.

The complete nucleotide sequence of human serum albumin mRNA has been determined from recombinant cDNA clones and from a primer-extended cDNA synthesis on the mRNA template. The sequence is composed of 2078 nucleotides, starting upstream from a potential ribosome binding site in the 5' untranslated region. It contains all the translated codons and extends into the poly(A) at the 3' terminus. Part of the translated sequence codes for a hydrophobic prepeptide, Met-Lys-Trp-Val-Thr-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr-Ser, followed by a basic propeptide, Arg-Gly-Val-Phe-Arg-Arg. These signal peptides are absent from mature normal serum albumin and, so far, have not been identified in their nascent state in humans. A remaining 1755 nucleotides of the translated mRNA sequence code for 585 amino acids, which are in agreement, with few exceptions, with the published amino acid sequence for human serum albumin. The mRNA sequence verifies and refines the repeating homology in the triple-domain structure of the serum albumin molecule.     

An intercistronic region and ribosome-binding site in bacterial messenger RNA.

A messenger RNA fragment about 220 nucleotides long has been isolated from 32-P-labeled tryptophan operon mRNA of Escherichia coli. When point mutations at the end of trpB and the beginning of trpA were introduced, the resulting nucleotide changes were found; hence the mRNA fragment must include the trpB-trpA intercistronic region. Most of the nucleotide sequences can be assigned to specific locations in the structural genes, based on the amino-acid sequences of the trpB and trpA proteins. In vitro, ribosomes bind to this piece of mRNA and protect from nuclease attack a region about 40 nucleotides long, containing a central AUG codon. The triplet codons to the 3' side of this AUG correspond to the first seven amino acids of the trpA protein; the codons to the 5' side correspond to the last six amino acids of the trpB protein. Translation of trpB is terminated by single UGA codon, which overlaps the trpA AUG initiation codon: UGAUG. Thus the untranslated "intercistronic" region consists of only two nucleotides. The RNA sequence spanning this region undoubtedly fulfills two functions, specifying ribosome recognition signals as well as encoding amino-acid sequences.     

Evidence for a human histone gene cluster containing H2B and H2A pseudogenes.

Not all members of the human histone gene family are functional. We have isolated a human H2B pseudogene that contains alterations in the protein-coding sequences as well as in the 3' and 5' flanking sequences that preclude expression of a functional H2B histone protein. There are three modifications in the amino acid-coding region: a single-base deletion producing a frame shift, a single-base substitution resulting in a codon change from serine to tryptophan (an amino acid not present in histones), and the absence of a stop codon. Analysis of nucleotide sequences upstream from the AUG start signal indicates the absence of a "TATA" box and other putative consensus regulatory sequences. In the 3' flanking region, a highly conserved block of 22 nucleotides that exhibits hyphenated dyad symmetry is displaced downstream. Within the same genomic segment, the adjacent H2A histone gene is missing 12 nucleotides, resulting in a deletion of four amino acids in a highly conserved region of the protein.