Tight linkage of retroviral-like sequences to a variant human c-mos gene in the human genome

Chumakov et al. [Gene 17 (1982) 19-26] identified in the human gene library a number of recombinant phages that possess a homology to the v-mos gene. Here we report the unusual structure of one of these recombinants, lambda gp5. The 14.3-kb stretch of human DNA from this phage contains at least three regions of homology to the v-mos gene, together with multiple copies of Alu-family repeats. Moreover, we have shown the presence of retrovirus-related sequences in the close vicinity of the mos-homologous regions. These data point to the possibility of involvement of retrovirus in the process of c-mos gene amplification during the formation of a multigene family.     

The nucleotide sequence of cDNA coding for preproinsulin from the primate Macaca fascicularis

DNA complementary to preproinsulin messenger RNA from the primate Macaca fascicularis has been cloned into the PstI endonuclease site of the plasmid pBR322. One clone contains the entire preproinsulin coding region as well as 59 nucleotides of the 5'-untranslated region. The results predict an amino acid sequence for the Macaca fascicularis preproinsulin and establish for the first time that the primary structures of human and primate insulins are identical. The two amino acid exchanges between human and primate preproinsulins are restricted to the pre- and the C-peptide, respectively.     

In vitro replication directed by a cloned adenovirus origin

A 5.7-kb recombinant plasmid, called XD-7, contains the terminal XbaI-E fragment from the left end of type 2 adenovirus cloned into the EcoRI site of pBR322. An average of 9% +/- 1% of input supercoiled, protein-free XD-7 DNA replicated as rolling circles with single-stranded tails ranging up to unit length and longer in reaction mixtures containing nuclear and cytoplasmic extracts from adenovirus-infected, but not uninfected, HeLa cells. The adenovirus origin was mapped on XD-7 by electron microscopy at the left boundary of the cloned adenovirus segment. Since replication proceeded rightwards, we conclude that the adenovirus l strand was displaced during replication. No origin was located at or near the EcoRI site on pBR322. Reversing the orientation of the adenovirus origin reversed the direction of replication, and deletion of the adenovirus origin abolished replication.     

Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.     

Mouse immunoglobulin A: nucleotide sequence of the structural gene for the alpha heavy chain derived from cloned cDNAs

The cDNAs complementary to mouse immunoglobulin alpha heavy chain mRNAs have been cloned into the PstI site of the plasmid vector pBR322. Recombinant plasmids have been identified by hybrid-arrested translation and purification of alpha heavy chain mRNA on DNA-DBM filters. The nucleotide sequence of the inserts encodes the constant and 3' untranslated regions of the alpha heavy chain mRNA. The CH3 domains of human and mouse alpha chains are highly homologous, including a 36 amino acid fragment not reported in the protein sequence (Robinson and Appella, 1980). As in the case of the mu secreted heavy chain, the alpha heavy chain contains a carboxy terminal piece of 20 amino acids.     

Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication

The 4150-bp plasmid pBR329 was constructed by the the insertion into pBR327 of an 877-bp DNA fragment carrying the Cmr gene from pBR328. This new cloning vector does not contain the 482-bp inverted duplication that has been reported to be present in pBR325 and pBR328 (Prentki et al., 1981). In pBR329 the Cmr gene lacks its original promoter but is transcribed counterclockwise toward the Apr gene by a promoter located to the right of the HindIII site in the Tcr gene.     

Cloning of alpha 2u globulin cDNA using a high efficiency technique for the cloning of trace messenger RNAs

An extremely high-efficiency technique is described for cloning double-stranded (ds) cDNAs in Escherichia coli. The method, which uses two synthetic oligonucleotide linkers rather than one, results in approx. 200--500 recombinant clones per ng of ds cDNA. This technique was used to clone a cDNA comprising 95% of the full length of the mRNA of alpha 2u globulin, a male rat liver protein, which represents approx. 1% of hepatic messenger RNA. The cloned probe was applied to study the complex hormone controls of alpha 2u globulin mRNA in male and female rats.     

Molecular cloning of AKR xenotropic murine leukemia virus unintegrated proviral DNA

Suprahelical proviral DNA of AKR xenotropic murine leukemia virus was purified from agarose gels and cloned in lambda Charon 28 DNA (BamHI sites). Nine viral DNA recombinants were identified and mapped with 12 restriction endonucleases. Three calsses of cloned viral DNA inserts were found: (1) Six inserts were apparently full-length 9.0-kb DNA with tandem long terminal repeat (LTR) elements; (2) two inserts contained DNAs with deletions in or adjacent to the LTR regions; (3) a single isolate contained an inversion of 2.3 kb around the LTR in the envelope gene.     

The human genome contains multiple sequences of varying homology to mouse mammary tumour virus DNA

Sequences related to the mouse mammary tumour virus (MuMTV) DNA were isolated from a genomic library of human DNA by screening under conditions of relaxed stringency. It is estimated that there are in the order of 50 MuMTV-like sequences per haploid genome and that the homology between the different human sequences and MuMTV varies by 15%.     

Comparison of the nucleotide sequence of cloned DNA coding for an apolipoprotein (apoVLDL-II) from avian blood and the amino acid sequence of an egg-yolk protein (apovitellenin I): equivalence of the two sequences

We have compared the amino acid sequences of two low-molecular-weight avian apoproteins: apoVLDL-II from very low-density lipoproteins of hen plasma and apovitellenin I from hen egg yolk. The sequence of White Leghorn apoVLDL-II was derived from the nucleotide sequence of cloned apoVLDL-II DNA (Chan et al., 1980). The sequenator was used to determine the amino acid sequence of apovitellinin I from two breeds of hen (White Leghorn and Australorp). The sequences from the two breeds were not only identical, but they also completely matched the predicted sequence derived from the apoVLDL-II DNA sequence. The identity reported here establishes that this protein is transported intact from the blood to the egg yolk.     

Cloning and characterization of a genomic DNA fragment carrying the basic copy of the gene coding for variant surface antigen 118 of Trypanosoma brucei

It has been proposed (Hoeijmakers et al., 1980b) that variant surface antigen (VSA) gene expression in Trypanosoma brucei is accomplished by a gene re-arrangement involving the basic copy of the VSA gene to give the so-called expression-linked copy (which is present only in the strain expressing that particular antigen). In this publication, the basic and expression-linked copies of the gene have been visualized by Southern blot analysis of nuclear DNA and shown to be located on HindIII fragments of 4.5 and 10-12 kb, respectively. In addition, several other bands of weaker hybridization are seen, probably representing evolutionary relatives. Using a shotgun approach, HindIII gene banks have been constructed and recombinants isolated which carry the 4.5-kb HindIII fragment containing the VSA118 gene basic copy. Several clones containing evolutionary relatives were also found. The 4.5-kb HindIII fragment is able to hybridize to probes derived from both the 5' and 3' ends of the cDNA, while the relatives have homology only to the 3' end. A detailed comparison of the restriction map of VSA118 cDNA with that of the VSA118 basic copy showed no differences, demonstrating that the gene contains no introns. This result also indicates that the gene from which VSA118 mRNA is transcribed (whether this be the basic copy or the expression-linked copy) is identical to the basic copy over the region analysed.     

Construction and characterization of a bacterial clone containing the hemagglutinin gene of the WSN strain (HON1) of influenza virus

A synthetic dodecadeoxynucleotide primer has been used to prepare a double-stranded DNA form of the hemagglutinin (HA) gene of a human influenza virus (WSN strain, HON1). This DNA has been inserted in plasmid pBR322 and cloned in bacterial cells. The insert contains nearly the complete hemagglutinin gene. A restriction map of this insert has been determined and structurally important areas of the HA gene have been sequenced. Amino acid sequences of several regions of the HA protein were deduced from the DNA sequences and compared to the known amino acid sequences of other influenza A viruses. WSN HA shows extensive homology to all influenza A viruses in a few regions, namely the first 17 amino acids of the N-terminus of HA1 (N-terminal polypeptide of HA) and the first 24 amino acids of the N-terminus of HA2 (C-terminal polypeptide of HA). The sequence diverges extensively from other influenza A viruses in most other areas. The sequence of WSN virus HA is similar to that of other HON1 viruses with the exception of the C-terminus of the HA1 peptide. The change in this area may contribute to some of the unique properties of WSN virus among the HON1 viruses. In addition, WSN HA contains a 17-amino-acid precursor before the N-terminus of HA1 and a single amino acid, arginine, connecting HA1 and HA2.     

A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates

DNA of the transforming, nondefective Epstein-Barr virus (EBV) strain M-ABA, which is derived from nasopharyngeal carcinoma cells, was cloned as large overlapping pieces into the cosmid pHC79. The termini were cloned from closed circular virus DNA molecules out of M-ABA cell DNA in phage λL47. The large overlapping clones were used to prepare a library of subclones with inserts of 1–15 kb. A detailed restriction enzyme map of M-ABA virus DNA reveals the close similarity to isolates from other sources. The high number of tandem repeats in EBV DNA stresses the importance of using cloning vectors that can be propagated in recA^?Escherichia coli hosts.