Single EcoRI Research Articles

Human Myeloperoxidase Gene: Molecular Cloning and Expression in Leukemic Cells

We have molecularly cloned the human myeloperoxidase (MPO) gene from the lambda gt11 expression library by screening with an affinity-purified MPO antibody. The cDNA clone of the MPO gene was used to study MPO gene expression in leukemic cells. The amino acid sequence predicted from the nucleotide sequence of the cDNA clone pMP401 matched exactly the 23 amino acid sequence of the NH2-terminal of the 60,000 MPO subunit. We found that MPO cDNA hybridized to a single EcoRI genomic band of 19 kb, indicating that the MPO gene represents a single gene in the human genome. Northern blot analysis of RNA isolated from leukemic cell lines and acute myelogenous leukemia (AML) patients' samples shows that MPO gene expression correlated with myeloid lineage. The intensity of MPO mRNA expression on Northern blot correlated with the level of MPO expression by cytochemical staining. Multiple species of MPO mRNA were found. This indicates that a single MPO gene may encode different RNA species through a mechanism of posttranscriptional processing or that multiple transcriptional start/termination sites exist in the MPO gene.

Human myeloperoxidase gene: molecular cloning and expression in leukemic cells

We have molecularly cloned the human myeloperoxidase (MPO) gene from the lambda gt11 expression library by screening with an affinity- purified MPO antibody. The cDNA clone of the MPO gene was used to study MPO gene expression in leukemic cells. The amino acid sequence predicted from the nucleotide sequence of the cDNA clone pMP401 matched exactly the 23 amino acid sequence of the NH2-terminal of the 60,000 MPO subunit. We found that MPO cDNA hybridized to a single EcoRI genomic band of 19 kb, indicating that the MPO gene represents a single gene in the human genome. Northern blot analysis of RNA isolated from leukemic cell lines and acute myelogenous leukemia (AML) patients' samples shows that MPO gene expression correlated with myeloid lineage. The intensity of MPO mRNA expression on Northern blot correlated with the level of MPO expression by cytochemical staining. Multiple species of MPO mRNA were found. This indicates that a single MPO gene may encode different RNA species through a mechanism of posttranscriptional processing or that multiple transcriptional start/termination sites exist in the MPO gene.

Myeloma mutant with a novel 3' flanking region: loss of normal sequence and insertion of repetitive elements leads to decreased transcription but normal processing of the alpha heavy-chain gene products.

We isolated and characterized LP1.2, a mouse myeloma mutant with a deletion of at least 4 kilobases (kb) immediately 3' of the alpha gene and introduction of at least 5 kb of novel (nonimmunoglobulin) sequence in its place. A 6.2-kb genomic EcoRI fragment from the mutated allele was cloned, and a subfragment was sequenced. The deletion begins 11 base pairs (bp) beyond the normal site of cleavage and polyadenylation for the secreted form of alpha mRNA. A short direct repeat, eight copies of the 17-mer GCCT ATAGAAGTAAGGA, is located at the junction of the alpha and novel sequences. The first 4 bp of the 17-mer are identical to the last 4 bp of the alpha sequence. Novel sequences downstream of the direct repeats in LP1.2 include a low-copy-number sequence flanked by two distinct, highly repetitive elements. The low-copy-number portion of the novel sequence appears on a single 30-kb EcoRI fragment in several myelomas and in liver DNA; one copy of this fragment has rearranged in cell line W3129, and this allele has rearranged a second time in LP1.2. LP1.2 contains low levels of apparently normal alpha protein and mRNA. The S1 nuclease protection of nuclear and cytoplasmic RNAs shows that cleavage and polyadenylation are efficient and accurate and that they occur without the accumulation of aberrant transcripts. Alpha transcription in isolated nuclei is decreased sevenfold in LP1.2 relative to its parent, which accounts for the low steady-state levels of cytoplasmic alpha mRNA and protein in LP1.2. Decreased alpha transcription could result either from the deletion of a positive regulator in the 3' flanking region or from the introduction of novel sequences which exert a negative effect.

Myeloma mutant with a novel 3' flanking region: loss of normal sequence and insertion of repetitive elements leads to decreased transcription but normal processing of the alpha heavy-chain gene products.

We isolated and characterized LP1.2, a mouse myeloma mutant with a deletion of at least 4 kilobases (kb) immediately 3' of the alpha gene and introduction of at least 5 kb of novel (nonimmunoglobulin) sequence in its place. A 6.2-kb genomic EcoRI fragment from the mutated allele was cloned, and a subfragment was sequenced. The deletion begins 11 base pairs (bp) beyond the normal site of cleavage and polyadenylation for the secreted form of alpha mRNA. A short direct repeat, eight copies of the 17-mer GCCT ATAGAAGTAAGGA, is located at the junction of the alpha and novel sequences. The first 4 bp of the 17-mer are identical to the last 4 bp of the alpha sequence. Novel sequences downstream of the direct repeats in LP1.2 include a low-copy-number sequence flanked by two distinct, highly repetitive elements. The low-copy-number portion of the novel sequence appears on a single 30-kb EcoRI fragment in several myelomas and in liver DNA; one copy of this fragment has rearranged in cell line W3129, and this allele has rearranged a second time in LP1.2. LP1.2 contains low levels of apparently normal alpha protein and mRNA. The S1 nuclease protection of nuclear and cytoplasmic RNAs shows that cleavage and polyadenylation are efficient and accurate and that they occur without the accumulation of aberrant transcripts. Alpha transcription in isolated nuclei is decreased sevenfold in LP1.2 relative to its parent, which accounts for the low steady-state levels of cytoplasmic alpha mRNA and protein in LP1.2. Decreased alpha transcription could result either from the deletion of a positive regulator in the 3' flanking region or from the introduction of novel sequences which exert a negative effect.

A map of the hCG beta-LH beta gene cluster.

The beta-subunit of human chorionic gonadotropin (hCG beta) is reportedly encoded by as many as seven non-allelic genes or pseudogenes. Previous studies have identified a cluster of three hCG beta gene copies and the single-copy lutropin-beta subunit (LH beta) gene, but overlap of this cluster with two additional pairs of hCG beta genes has not been demonstrated, despite the isolation of 18 genomic clones. To define the number and organization of non-allelic hCG beta gene copies, genomic Southern blot analyses were performed using hCG beta cDNA and gene-flanking unique-sequence probes. The data show the linear arrangement of six genes (or pseudogenes) and show that the hCG beta-IH beta gene cluster is present in a single 58-kilobase EcoRI fragment. Our map of the cluster indicates which of the cloned hCG beta genes reflect somatic genotypes rather than recombinant artifacts and will thus permit investigation of factors regulating expression during gestation.

Endonuclease VII resolves Y-junctions in branched DNA in vitro.

Endonuclease VII (gp 49 of phage T4) resolves four-way junctions in branched DNAs. We have extended our investigations of the specificity of endo VII and tested its activity with three-way junctions (Y-structures) constructed in vitro. Both 'closed' and 'open' Y-structures were made, absolutely identical in sequence but differing from each other by a single nick in one of the three arms. Pure Y-structures were obtained on a preparative scale by annealing plus and minus strands from two M13mp strains. One strain has an inverted repeat of 2 X 31 nucleotides cloned into the single EcoRI site while in the other strain this repeat is absent. The structures were used in reactions with endo VII, which recognizes the branch point of both structures and introduces a characteristic number of nicks, 3' to the junction in each arm of the structure. Strong and weak sites could be distinguished and the cleavage pattern differed significantly between the two structures. The observed resolution of Y-junctions by endo VII in vitro is compatible with a model for the resolution of recombinant Y-branches in DNA.

Tn 1721 derivatives for transposon mutagenesis, restriction mapping and nucleotide sequence analysis

New derivatives of the tetracycline-resistance transposon Tn 1721 that carry resistances to chloramphenicol, tetracycline, kanamycin and streptomycin are described. These elements are provided on various plasmid vehicles and as chromosomal insertions to extend the range of targets for Tn mutagenesis. Single EcoRI sites at the ends of these transposons proved most useful (i) for physical mapping, (ii) for the generation of new EcoRI sites in cloning experiments, (iii) for end-labelling and (iv) for sequencing of DNA adjacent to an insertion.

Characterization of the gene encoding the major rat liver asialoglycoprotein receptor.

A cloned cDNA encoding the major rat liver asialoglycoprotein receptor has been used to analyze the gene for this protein. Genomic Southern blot analysis reveals that the gene is contained on a single EcoRI restriction fragment and is unique. A clone containing the gene (isolated from a rat liver genomic library) has been characterized by sequence analysis. The mRNA for the receptor is encoded by nine exons separated by eight introns. The first exon is confined to the 5'-untranslated region of the mRNA, the second exon encodes most of the cytoplasmic NH2-terminal domain of the receptor polypeptide, the third exon corresponds to the hydrophobic transmembrane portion of the polypeptide, and the remaining exons encode the extracellular parts of the receptor. Some, but not all, of the divisions between exons correspond to boundaries between functional domains of the polypeptide.

Molecular cloning of a Bacillus subtilis gene involved in spore outgrowth.

A lambda Charon 4A derivative carrying the outB gene of Bacillus subtilis has been identified by transformation of a B. subtilis mutant temperature-sensitive in spore outgrowth. The cloned region is a single EcoRI fragment 14 kb in length. In addition to outB, the cloned DNA includes at least part of the amyE and aroI loci.

Heterologous hybridization of bacterial DNA to the endoglucanases A and B structural genes celA and celB of Clostridium thermocellum

DNA from various cellulolytic and non-cellulolytic bacteria was found to hybridize to Clostridium thermocellum NCIB10682 DNA fragments carrying the structural genes celA and celB which code for endoglucanases A and B. Homology to celA was detected in Agrobacterium rhizogenes, Azospirillum brasilense, Bacillus subtilis, Cellulomonas sp., Clostridium stercorarium, Erwinia chrysanthemi, Pseudomonas solanacearum and Streptomyces griseus. Homology to celB was detected only in B. subtilis, C. stercorarium and E. chrysanthemi. No homology to celA or celB was detected in Agrobacterium tumefaciens, Rhizobium japonicum, Rhizobium meliloti, Rhizobium ORS571 and Trichoderma reesei. Hybridization performed with the homologous strain NCIB10682 and with C. thermocellum LQRI suggested that the two strains were identical in terms of cel genes. In addition, it is likely that the C. thermocellum cel genes, whose number is presently estimated to be at least ten, do not belong to a gene family, since heterologous hybridization was observed only to a single small EcoRI or HindIII fragment homologous to celB.

Construction of a Gene Bank of Streptococcus lactis Plasmid pLM2001 Deoxyribonucleic Acid in Escherichia coli

Plasmid pLM2001, which is essential for the fermentation of lactose by Streptococcus lactis LM0232, was isolated and cut into eight fragments with restriction endonuclease EcoRI. The deoxyribonucleic acid fragments ranged in size from approximately .8 to 10.3 kilobases as determined by agarose gel electrophoresis. EcoRI-restricted pLM-2001 deoxyribonucleic acid was shotgun cloned into the single EcoRI site of the multifunctional Escherichia coli-Streptococcus sanguis shuttle vector, pSA4, insertionally inactivating the chloroamphenicol resistance gene. Following transformation of the deoxyribonucleic acid into Escherichia coli HB101, 242 transformants containing chimeric plasmids were detected by their resistance to tetracycline and failure to grow in the presence of chloramphenicol. Plasma deoxyribonucleic acid from 98 transformants was cleaved with EcoRI and analyzed by agarose gel electrophoresis for the presence of pLM2001 inserts. Clones were obtained, which individually contained each of the eight fragments. In addition, a series of clones containing various combinations of the fragments was obtained. The availability of a system for cloning and rapidly screening for fragments of Streptococcus lactis deoxyribonucleic acid under conditions where deletion of passenger or vector deoxyribonucleic acid does not occur will facilitate studies designed to isolate and characterize metabolically important genes carried by plasmids from the group N streptococci.

Comparison of the transposon-like structures encoding clindamycin resistance in Bacteroides R-plasmids

The R-plasmids pBF4, pBFTM10, and pBI136 encode transmissible clindamycin resistance (Cc r) in Bacteroides spp. These plasmids are distinct replicons but the regions implicated in Cc r share some homology and appear to have a transposon-like structure. To better understand the mechanism of dissemination and to locate the Cc r determinant(s), the genetic and structural properties of the Cc r regions of each plasmid were compared and contrasted. For this work a single EcoRI restriction fragment containing the Cc r region from each plasmid was cloned into pBR322 in Escherichia coli. Results of restriction mapping and heteroduplex experiments showed that the pBF4 EcoRI-D and pBFTM10 EcoRI-B fragments shared more than 90% base sequence homology but that the EcoRI-C fragment of pBI136 had diverged significantly. The pBI136 fragment also did not confer tetracycline resistance in E. coli as shown for the pBF4 EcoRI-D fragment (D. G. Guiney, P. Hasegawa, and C. E. Davis, 1984, Plasmid 11, 248–252). Heteroduplex experiments showed that the pBI136 EcoRI-C and pBF4 EcoRI-D fragments shared a 1.2-kb region of homology attributed to a directly repeated sequence which bounds the Cc r region. Southern hybridization studies indicated that an additional 0.85 kb of the pBI136 EcoRI-C fragment was homologous to the EcoRI-D fragment of pBF4. This region was characterized by its sequential restriction endonuclease sites for HindIII, AvaII, and DdeI, and it is proposed that the Cc r gene(s) resides in this area.

Expression in Escherichia coli of streptococcal plasmid-determined erythromycin resistance directed by the cat gene promoter of pACYC 184.

The streptococcal erythromycin resistance (Emr) plasmid pSM7 (6.4 kb) and the E. coli vector pACYC184 (4.0 kb) were fused at their single EcoRI sites to form the bifunctional chimeric plasmid pSM7184 (10.4 kb) in which the Emr determinant was placed under control of the chloramphenicol acetyl transferase (cat) promoter of pACYC184. In the sense orientation (orientation I) of pSM7, the cat promoter directed expression of Emr in the E. coli host strains 294 and DB11 more efficiently than did the indigenous transcription signals of pSM7, which were functional in the opposite orientation II. In Streptococcus sanguis (Challis), the level of Emr was independent of the orientation of pSM7 in pACYC184, showing that the cat promoter was not recognized in the gram-positive host. The growth of E. coli (pSM7184I) in a defined medium containing glycerol as carbon source, or containing glucose plus extraneous cyclic 3'-5' adenosine monophosphate (cAMP) led to an Emr level which was 15-30 times higher than that of cultures grown on glucose. These results showed that under control of the cat promoter, Emr is subject to cAMP-mediated catabolite repression and provided conclusive evidence that the enhancement of Emr expression in E. coli carrying pSM7184I is controlled at the transcriptional level. Besides enabling us to determine the orientation of transcription of the Emr gene in pSM7 and related vectors, this work also made available new bifunctional cloning vehicles able to replicate in both E. coli and S. sanguis.

Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster

For insertional mutagenesis of Pseudomonas aeruginosa, a derivative of the kanamycin-resistance (Km R) transposon Tn5 was constructed (Tn 5-751) that carried the trimethoprim-resistance (Tp R) determinant from plasmid R 751 as an additional marker. Double selection for Km R and Tp R avoided the isolation of spontaneous aminoglycoside-resistant mutants which occur at high frequencies in P. aeruginosa. As a delivery system for the recombinant transposon, plasmid pME305, a derivative of the broad-host-range plasmid RP1, proved effective; pME305 is temperature-sensitive at 43°C for maintenance in Escherichia coli and P. aeruginosa and deleted for IS 21 and the Km R and primase genes. In matings with an E. coli donor carrying pME9 (= pME305::Tn5-751), transposon insertion mutants of P. aeruginosa PAO were recovered at approx. 5 × 10 −7/donor at 43°C. Among Tn 5-751 insertional mutants 0.9% were auxotrophs. A thr::Tn5-751 mutation near the recA-like locus rec-102 is useful for the construction of recombination-deficient strains. Several arc::Tn5-751 mutants could be isolated that were defective in anaerobic utilization of arginine as an energy source. From three of these mutants the arc gene region was cloned into an E. coli vector plasmid. Since Tn 5-751 has a single EcoRI site between the Tp R and Km R genes, EcoRI-generated fragments carrying either resistance determinant plus adjacent chromosomal DNA could be selected separately in E. coli. Thus, a restriction map of the arc region was constructed and verified by hybridization experiments. The arc genes were tightly clustered, confirming earlier genetic evidence.

Molecular cloning of AIDS-associated retrovirus.

Retroviruses cause a wide variety of diseases in avian and mammalian species. Human acquired immune deficiency syndrome (AIDS) leads to collapse of the immune system and death by a wide variety of opportunistic infections; unusual forms of cancer are associated with this syndrome. Retroviruses have been recovered from tissues of AIDS patients and from patients with related conditions. These similar newly-isolated viruses are lymphadenopathy-associated virus (LAV), human T-cell lymphotropic virus (HTLV-III) and AIDS-associated retrovirus (ARV-2). We have identified a RNA genome of approximately 9 kilobases (kb) in virions purified from the culture medium of a human T-cell tumour line infected with ARV-2. A cDNA probe made from viral RNA detected circular DNA molecules and proviral forms in infected cells. We prepared a library of infected cell DNA. Recombinant phage included those with a 9.5-kb proviral DNA and viral DNA permuted with respect to the single EcoRI site. Comparison of three ARV isolates from different AIDS patients revealed polymorphism of restriction endonuclease sites.

Characterisation of the ColE8 plasmid, a new member of the group E colicin plasmids

We have determined the restriction map of the ColE8-J plasmid after cloning it into the pBR322 vector. By subcloning and transposon mutagenesis we have localized the colicin immunity gene, the colicin structural gene, and lys, the region that determines MC sensitivity. In contrast to the ColE3-CA38 plasmid, the genes coding for colicin E8 production and immunity cannot be cloned on a single EcoRI fragment. Insertion of Tn5 transposons into the colicin structural gene region of the recombinant plasmid inactivated colicin production and MC sensitivity. Insertion of transposons into the lys region reduced colicin E8 production and MC induced lysis, the extent of which was dependent upon the precise site of insertion. We propose that the colicin E8 structural gene and lys must be transcribed from a common promoter situated proximal to the structural gene, whilst the colicin E8 immunity gene is transcribed from a second promoter. The lys region is responsible both for cell lysis after MC induction and positive regulation of colicin E8 synthesis.

FBR murine osteosarcoma virus I. Molecular analysis and characterization of a 75,000-Da gag-fos fusion product

The FBR murine osteosarcoma virus complex induces bone tumors with a similar latency and pathology to those induced by the FBJ virus complex. FBR murine sarcoma virus (FBR-MSV) has been isolated from its helper virus(es) by the establishment of transformed nonproducer cells. These cells were found to express a 75,000-Da protein (P75) which was antigenically related to the p55 oncogene product of the FBJ murine osteosarcoma virus (FBJ-MSV). P75 also contained antigenic determinants of murine leukemia virus (MLV) gag gene p15, p12, and p30 proteins, and is therefore a gag-fos fusion protein (P75 gag-fos ). P75 gag-fos is a phosphoprotein and is found primarily in the nucleus. Only a single species of RNA, of 3.3 kb, was identified in FBR-MSV-transformed nonproducer cells using both fos and MLV probes, which suggested that P75 gag-fos was expressed from genome-sized RNA. Chromosomal DNA from one nonproducer cell line was found to contain a single EcoRI restriction fragment of 12 kb pairs (kbp) which encompassed the FBR-MSV provirus. This DNA fragment was molecularly cloned into bacteriophage Charon 30 (λFBR-1), and a 7.5-kbp HindIII restriction fragment containing the entire provirus was subsequently subcloned into pBR322 (pFBR-1). DNA from pFBR-1 was capable of inducing morphological transformation of mouse and rat fibroblasts in tissue culture. In addition, transfected cells expressed the FBR-MSV P75 gag-fos protein.

Nucleotide sequence of cloned cDNA coding for honeybee prepromelittin.

Total mRNA from venom glands of young queen bees was transcribed into cDNA and cloned into the PstI site of plasmid pBR322. The nucleotide sequence of two clones with inserts containing genetic information for prepromelittin is presented. The longer insert encompasses 374 base pairs including 52 nucleotides before the initiation codon and a 3' non-coding region of 112 base pairs. The 70 amino acids of prepromelittin represent the total coding capacity of the mRNA from which this insert is derived. Southern blot analysis with this cloned cDNA has shown that it hybridizes with a single EcoRI fragment of honeybee DNA which contains about 3000 base pairs.

Methyl-directed repair of DNA base-pair mismatches in vitro.

An assay has been developed that permits analysis of DNA mismatch repair in cell-free extracts of Escherichia coli. The method relies on repair of heteroduplex molecules of f1 R229 DNA, which contain a base-pair mismatch within the single EcoRI site of the molecule. As observed with mismatch heteroduplexes of lambda DNA [Pukkila, P. J., Peterson, J., Herman, G., Modrich, P. & Meselson, M. (1983) Genetics, in press], in vivo mismatch correction of f1 heteroduplexes is directed by the state of dam methylation of d(G-A-T-C) sequences within the DNA duplex. Thus, the heteroduplex (formula: see book) is repaired in vivo to an EcoRI-sensitive form if the strand bearing the wild-type EcoRI sequence carries the dam modification and the other does not. Such molecules are also subject to mismatch repair by E. coli extracts. The in vitro activity is also dependent on ATP, the state of dam methylation of mismatch heteroduplexes, and products of mutH, mutL, mutS, and uvrE loci. However, crude fractions deficient in these gene products do complement in the cell-free system, thus providing assays for their isolation. The in vitro reaction is accompanied by repair synthesis on the unmethylated DNA strand.

Structure of the hepatitis B virus genome.

The extent and position of the single-stranded gap in DNA molecules from Dane particles isolated from two donors of the adw serotype were determined by molecular hybridization and electron microscopic methods. The results showed that in each preparation more than 99% of the circular molecules are of uniform length and contain both single- and double-stranded regions. They confirmed that one end of the short strand is fixed with respect to the single EcoRI site within the molecule and to the nick in the long strand, but they also showed that although the position of the other end is variable, there is a preferred minimum length of about 650 to 700 nucleotides for the single-stranded region.