Cloning of the DNA BglII fragments of bacteriophage T4 in the vector plasmid pSCC31

An attempt has been made to clone six BglII fragments of T4 DNA in the range of 3.3-8.1 kb in the vector plasmid pSCC31 containing a single BglII site within the gene for endonuclease EcoRI and pL promoter of phage lambda. DNA fragments were extracted from the corresponding bands of agarose gel. The following BglII fragments were cloned: the 3.3 kb fragment No. 9 containing a portion of gene 20, the gene 21 and a portion of gene 22; the 4.2 kb fragment No. 8.1 with genes 17, 18, 19 and a portion of gene 20; the 5.2 kb fragment No. 7.1 with genes 25-29 and a portion of gene 48. In the case of the fragment No. 7.1, the recombinant plasmids pRL705 and pRL707 with different orientation of phage DNA fragment were obtained. An attempt to clone the fragments No. 8.2 (4.2 kb), No. 7.2 (5.45 kb) and No. 6 (8.1 kb) was unsuccessful and this probably indicates the presence of the genes, whose products are deleterious to the growth of bacterial cell.     

Isolation of the chicken beta-globin gene and a linked embryonic beta-like globin gene from a chicken DNA recombinant library.

A library of random chicken DNA fragments, 15-22 kb long, has been prepared in the vector lambda Charon 4A. This library was screened with combined adult and embryonic globin cDNA, and several independent globin gene-containing recombinants were isolated. One of these recombinants, lambda Chicken beta-globin 1 (lambda C beta G1), contains the adult chicken beta-globin gene and a closely linked embryonic beta-like globin gene. Both genes are transcribed in the same direction with the adult gene located 5' to the embryonic gene. Electron microscopic visualization of R loop structures generated by hybridization of globin RNA to lambda C beta G1 demonstrates that both globin genes contain major intervening sequences about 800 bp long, similar to those present in mammalian beta-globin genes. The adult beta-globin gene also contains a minor (approximately 100 bp long) intervening sequence analogous to the one observed in mammalian beta-globin genes. Restriction enzyme analysis of the adult beta-globin gene on lambda C beta G1 is consistent with the hypothesis that its two intervening sequences occur in the same positions with respect to the beta-globin amino acid sequence as do the corresponding mammalian intervening sequences.     

A method of identifying and isolating a unique member of a multigene family: application to a trypanosome surface antigen gene.

A chimeric oligonucleotide was constructed using DNA sequences from two distal regions of a cDNA which encodes a major surface antigen (TSA-1) of Trypanosoma cruzi. Conditions were found that allowed the chimeric oligonucleotide to hybridize only to a 5.4 kb EcoRI fragment in a Southern blot of total genomic DNA. The 5.4 kb EcoRI genomic DNA fragment has previously been shown to be located at a telomeric site, thus the studies described here directly demonstrate that the TSA-1 gene is telomeric in location. It is also shown that the chimeric oligonucleotide can be used to selectively identify recombinant lambda phage which harbor the TSA-1 gene using standard library screening procedures. Since these studies demonstrate that a chimeric oligonucleotide can be used to identify in both Southern blots and library screens a single member among the more than sixty members of the TSA-1 gene family, it seems likely that chimeric oligonucleotides may be of general use in studies involving repetitive DNA sequence families.     

用富集文库克隆人胰岛素基因组基因

通过构建可富集人胰岛素基因的λ噬菌体文库,克隆了人胰岛素基因组基因．首先从中国人血液白细胞中提取到人基因组ＤＮＡ,用ＥｃｏＲⅠ和ＢｇｌⅡ对基因组ＤＮＡ进行全酶切,经０．４％琼脂糖凝胶电泳,特异回收９．５ｋｂ左右的ＤＮＡ片段．将该片段与λＥＭＢＬ３／ＢａｍＨⅠ臂连接,构建成一个特殊的人基因组λ噬菌体文库（富集文库）,效价为２×１０４．同时采用ＰＣＲ方法及用引物Ⅰ：５′ＧＧＡＣＡＧＧＣＴＡＣＡＴＣＡＧＧＡＡＧＡＧＧ３′,引物Ⅱ：５′ＣＴＧＣＧＴＣＴＡＡＴＴＧＣＡＧＴＡＧＴＴＣ３′,从人基因组ＤＮＡ中扩增出一段含胰岛素基因的１．３６ｋｂＤＮＡ片段,做为放射性标记探针,对文库进行了噬菌斑原位杂交筛选,从１×１０４个噬菌斑中筛选到一个含人胰岛素基因组基因的阳性克隆,并进一步完成了亚克隆和该基因１７３２ｂｐＤＮＡ序列的测定．结果该基因的１７３２ｂｐＤＮＡ序列包括部分５′端和３′端与国外发表的人胰岛素α型等位基因的序列相同     

Cloning and expression of a mouse adenine phosphoribosyltransferase gene

A functional mouse adenine phosphoribosyltransferase (APRT) gene was identified and cloned by screening a mouse sperm genomic DNA library in lambda Charon 4A. The probe utilized for screening was a restriction fragment encoding much of the hamster APRT gene. Six recombinants that hybridized with the probe were identified, and after digestion with restriction enzymes EcoRI and PvuII revealed three different patterns of digestion for each enzyme. Of the six recombinants, five representing two of the restriction patterns possessed transforming activity. A sixth recombinant, which has a unique restriction pattern, lacks transforming activity but hybridizes well with hamster APRT coding sequences and is a possible candidate for a pseudogene. We used three criteria for conclusively identifying the mouse APRT genes. (1) DNA from the recombinant lambda phage hybridizes with DNA encoding hamster APRT. (2) The recombinant lambda phages and their DNAs transform mouse, hamster and human APRT- cells to the APRT+ phenotype. (3) The hamster and human transformants display APRT activity that migrates with a mobility characteristic of mouse APRT and not of hamster or human. A 3.1-kb EcoRI-SphI restriction fragment which retains transforming activity has been subcloned into the plasmid pBR328. Comparison of restriction enzyme sites with those contained in a mouse APRT cDNA, coupled with loss of transforming activity after enzyme digestion, indicates that the mouse APRT gene is larger than 1.8 kb and contains at least three introns.     

Primary structure of an EcoRI fragment of lambda imm434 DNA containing regions cI-cro of phage 434 and cII-o of phage lambda.

Digestion of phage lambda imm434 DNA with restriction endonuclease EcoRI yields 7 fragments. The shortest among them (1287 bp) contains the right part of the phage 434 immunity region and the phage DNA portion proximal to it. The complete primary structure of this fragment has been determined using the chemical method of DNA sequencing. Hypothetical amino-acid sequences of proteins coded by the cro gene of phage 434 and the cII gene of phage lambda, as well as NH2-terminal amino-acid sequences of the cI protein of phage 434 and the O protein of phage lambda, have been deduced solely on the basis of the DNA sequence. The fragment studied contains also the pR and probably prm promoters and the oR operator of phage 434. The sequence coding for them differs from the respective DNA sequence of phage lambda.     

Tn5cos: a transposon for restriction mapping of large plasmids using phage lambda terminase.

A method for the rapid restriction mapping of large plasmids has been developed. A 400-bp fragment of phage lambda DNA containing the cos region has been inserted into Tn5. After in vivo transposition of this Tn5cos element into the plasmid of choice, the plasmid is isolated and linearized at its cos site with phage lambda terminase (Ter). Such Ter linearization was about 70% efficient. After partial digestion of the linear molecules with the appropriate restriction enzyme, the products are selectively labelled at the right or left cohesive phage lambda DNA termini by hybridization with digoxygenin (DIG)-11-dUTP-labelled (using terminal transferase) oligodeoxyribonucleotides complementary to the single-stranded cos ends. After pulsed field gel electrophoresis, the labelled fragments are visualized in the dried gel using a DIG-detection kit. The restriction map can be directly determined from the 'ladder' of partial digestion products.     

Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping.

Tn5-tagged invasion plasmid DNA (pWR110) from Shigella flexneri serotype 5 (strain M90T) was cloned into the expression vector lambda gt11. Recombinant phage (lambda gt11Sfl) expressing pWR110-encoded polypeptide antigens were identified by using rabbit antisera directed against S. flexneri M90T invasion plasmid antigens. Antigens encoded by lambda gt11Sfl recombinant phage were characterized by reacting affinity-purified antibodies, eluted from nitrocellulose-bound plaques of lambda gt11Sfl recombinants, with virulent, wild-type S. flexneri M90T polypeptides in Western blot analyses. lambda gt11Sfl clones directing the synthesis of complete, truncated, and beta-galactosidase fusion versions of three previously identified outer membrane polypeptides (57-, 43-, and 39-kilodalton [kDa] antigens) were isolated. A fourth polypeptide, similar in size to the 57-kDa antigen (ca. 58 kDa) but unrelated as determined by DNA homology and serological measurements, was also identified. Southern blot analysis of S. flexneri M90T invasion plasmid DNA hybridized with lambda gt11Sfl insert DNA probes was used to construct a map of invasion plasmid antigen genes (ipa) corresponding to the 57-kDa (ipaB), 43-kDa (ipaC), and 39-kDa (ipaD) polypeptides. Genes ipaB, ipaC and ipaD mapped to contiguous 4.6-kilobase (kb) and 1.0-kb HindIII fragments contained within a larger (23-kb) BamHI fragment. The ipaH gene, which encodes the synthesis of the 58-kDa polypeptide, did not map in or near the ipaBCD gene cluster, suggesting a distinct location of ipaH on the invasion plasmid.     

Structure and expression of human globin genes introduced into mouse fibroblasts

The human delta- and beta-globin genes, contained in a recombinant bacteriophage (lambda H beta G1), were introduced into mouse fibroblasts by cotransformation with a plasmid (chi 1) containing the herpes simplex thymidine kinase gene using the calcium phosphate precipitation technique. A molar ratio of lambda H eta G1 to chi 1 DNA of 3:1 was used. Four of the eleven stable transformants obtained contained intact delta- and beta-globin genes as determined by Southern blot analysis. To assess methylation in the segment of human DNA introduced into mouse cells, digestion with Hpa II or Msp I alone or with a second restriction enzyme was performed. The sites examined near the human delta- and beta-globin genes in transformed cells were not methylated. RNA extracted from the transformed cells was analyzed by RNA-cDNA hybridization; no more than 100 copies of human beta-globin mRNA/cell were found. Although hypomethylation of sites surrounding expressed globin genes in erythroid cells has been described, this property is not sufficient to ensure a high level of expression in fibroblasts.     

Isolation and characterization of the complete chicken beta-globin gene region: frequent deletion of the adult beta-globin genes in lambda.

A library of bacteriophage lambda clones containing chicken chromosomal DNA was screened, using the adult beta-globin cDNA plasmid pHb 1001 as a probe. Sixteen overlapping clones were isolated containing 35 kilobase pairs (kbp) of chicken DNA. Characterization of these clones revealed four beta-like globin genes, some genomically repeated sequences, but no pseudo-genes. The four beta-like genes have an average intergenic distance of less than half of that found for the mammalian beta-like globin gene clusters so far characterized. The overall features of the map were confirmed by genomic Southern analysis. Frequent deletions were shown to occur between the various beta-like globin genes during phage propagation. The presumptive hatching gene in particular was always associated with abnormal lambda clones although we were able to find one such clone that did contain a normal copy of the hatching gene itself. Probably such deletions explain the failure to recover this gene in previous attempts.     

ColE1 cloning of a ribosomal RNA promoter region from lambdarifd18 by selection for lambda integration and excision functions.

The expression of the ribosomal RNA gene carried by the lambda transducing phage lambdarifd18 is shown to be subject to stringent amino acid control. lambdarifd18 DNA was digested with endonuclease EcoRI and ligated to similarly restricted ColE1 plasmid DNA. Selection for expression of lambda integration and excision gene activity carried by the same DNA fragment results in cloning of the promoter proximal portion of the 16S ribosomal RNA gene. The resulting chemera expresses lambda integration and excision functions as well as encoding the promoter proximal half of a 16S ribosomal RNA gene.     

Cloning and mapping of infA, the gene for protein synthesis initiation factor IF1.

The gene for translation initiation factor IF1, infA, has been identified by using two synthetic oligonucleotides to screen a Charon 30 library of Escherichia coli DNA. A recombinant lambda phage, C1921, was purified from a plaque positive for both probes. A 2.8 kb BglII fragment and a 2.0 kb HindIII fragment isolated from C1921 were subcloned into the BamHI and HindIII sites of pBR322 to yield pTB7 and pTH2 respectively. Synthesis of IF1 in maxicells transformed with pTB7 or pTH2 indicates the presence of inf A in both inserts. This was confirmed by DNA sequencing: a region was found that codes for a 8,119 dalton protein with an amino acid sequence corresponding to IF1. The chromosomal location of inf A was determined by mapping the closely linked beta-lactamase gene (Ampr) in pTB7 and pTH2. pTB7 and pTH2 were transformed into polA Hfr hosts, and integration of the plasmid by homologous recombination near inf A was selected on the basis of ampicillin resistance. The site of integration was confirmed by Southern blot analysis of restriction nuclease digested wild type and transformed genomic DNA. The Ampr marker (and therefore inf A) was mapped to about 20 minutes by Hfr interrupted matings and P1 transduction experiments. The structure and regulation of the inf A operon currently are being investigated.     

Cloning and physical mapping of DNA sequences encompassing a region in N-myc amplicons of a human neuroblastoma cell line.

Cloning and physical mapping of DNA sequences encompassing N-myc amplicons of a human neuroblastoma cell line were done. A number of lambda phage clones within this region were isolated using the probes prepared by the phenol emulsion reassociation technique. Based on the restriction mapping, they were integrated into 8 contigs with sizes of 25-60 kb which, in total, encompassed a 330 kb region. Several amplicons, 100, 420, 480 and 520 kb in size as a Notl fragment, were identified using hexagonal field gel electrophoresis, and the contigs were assigned in these Notl fragments. The region encompassed by the contigs was equivalent to some 60-80% of the amplicons identified as a Notl fragment. In order to compare the amplified regions flanking the N-myc gene among the cell lines, the phage clones to cover the whole contigs were used for hybridization as a probe. The results showed that the portions of the whole contigs ranging 18-45% were also amplified in the cell lines examined. These results allowed us to identified the 'rearranged sites' which were rather evenly distributed, one at every 40 kb, through the contigs. These observations lead to the idea that an amplified DNA domain is constructed after the multiple rearrangements and then increases in number, finally resulting in the formation of subsets of amplicons with sequence homogeneity.     

Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nanogram quantities of DNA

We have been developing a rapid and convenient assay for the measurement of DNA damage and repair in specific genes using quantitative polymerase chain reaction (QPCR) methodology. Since the sensitivity of this assay is limited to the size of the DNA amplification fragment, conditions have been found for the quantitative generation of PCR fragments from human genomic DNA in the range of 6-24 kb in length. These fragments include: (1) a 16.2 kb product from the mitochondrial genome; (2) 6.2, 10.4 kb, and 15.4 kb products from the hprt gene, and (3) 13.5, 17.7, 24.2 kb products from the human beta-globin gene cluster. Exposure of SV40 transformed human fibroblasts to increasing fluences of ultraviolet light (UV) resulted in the linear production of photoproducts with 10 J/m(2) of UVC producing 0.085 and 0.079 lesions/kb in the hprt gene and the beta-globin gene cluster, respectively. Kinetic analysis of repair following 10 J/m(2) of UVC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt gene and beta-globin gene cluster was 7.8 and 24.2 h, respectively. Studies using lymphoblastoid cell lines show very little repair in XPA cells in both the hprt gene and beta-globin locus. Preferential repair in the hprt gene was detected in XPC cells. Cisplatin lesions were also detected using this method and showed slower rates of repair than UV-induced photoproducts. These data indicate that the use of long targets in the gene-specific QPCR assay allows the measurement of biologically relevant lesion frequencies in 5-30 ng of genomic DNA. This assay will be useful for the measurement of human exposure to genotoxic agents and the determination of human repair capacity.     

A new host-vector system allowing selection for foreign DNA inserts in bacteriophage lambda gtWES.

An improved vector (lambda gtWES.T5-622) for EcoRI fragments has been derived from EK2 vector lambda gtWES.lambdaB' by replacing the lambda B fragment with two identical 1.1 Md fragments from the pre-early region of bacteriophage T5. The new vector has two advantages which facilitate elimination of parental-type recombinants in an in vitro recombination experiment. Firstly, the 1.1 Md insert is too small to be re-inserted into lambda gtWES in a single copy. Secondly the 1.1 Md T5 fragment carries T5 gene A3 which prevents growth of phage retaining this fragment when the Excherichia coli host carries plasmid ColIb. Thus, essentially all plaques are due to phage with donor DNA inserts and are free of T5 DNA fragments. The size usually given as the theoretical minimum size for insertion into the lambda gt series of vectors is 0.66 Md. We have shown that this size is an underestimate and that the lower limit is about 1.6 Md. A precise estimate is difficult since there is strong selection, among phage having small inserts, for those which have acquired additional genetic material by duplication of the lambda DNA.     

A vehicle for DNA transfer and for recovery of transferred genes: lambda Charon phage-pBR322 hybrid

Recombinant Charon 4A phages accommodating the Herpes simplex virus (HSV-1) thymidine kinase (tk) gene, the ampicillin-resistance (ApR) gene, and the replication origin of pBR322 were constructed. The phage DNA was introduced into mouse Ltk- cells by a free DNA transfer method or phage-mediated DNA transfer method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607]. Analyses of the physical state of the transferred DNA in the recipient cell genome showed that a DNA fragment as long as 12.7 kb was integrated intact into 67% and less than 40% of the Ltk- transformant cells by phage-mediated DNA transfer and by free DNA transfer, respectively. We also developed a new rapid method for recovery of the transferred gene from the Ltk+ cell into Escherichia coli; the method depends on the fact that the recombinant lambda phage carrying the ApR gene and replication origin of pBR322 transduces lambda-lysogenic bacteria to ApR and is maintained as a plasmid. Using this method the HSV-1 tk gene from one Ltk+ transformant was rapidly and successfully recovered without any rearrangement of the target sequence.     

Construction of a hybrid bacteriophage-plasmid recombinant DNA vector.

A phage-plasmid hybrid was constructed for use as a recombinant DNA vector, allowing the propagation of cloned EcoRI restriction endonuclease fragments of about 2 X 10(6) to 11 X 10(6) daltons. The colicin E1 plasmid replicon was fused to the left arm of a lambdagt generalized transducing phage with a thermolabile repressor, yielding a genome which could be replicated either by phage lambda functions or via the colicin E1 plasmid replicon. At the nonpermissive temperature, phage functions were derepressed and phage growth occurred lytically. Alternatively, at the permissive temperature, lambda functions were repressed and the vector replicated as a covalently closed circular plasmid. The phage-plasmid hybrid vector could be maintained at a copy number determined by the colicin E1 plasmid replicon and was also sensitive to amplification after chloramphenicol treatment. An EcoRI fragment of Escherichia coli DNA encoding genes of the arabinose operon also was inserted into the central portion of the vector.     

Phasmids. Properties and the use in genetic engineering. I. Construction of the phasmid vector

A phasmid vector molecule designated pMYF11 has been constructed. The vector combines some useful features of plasmid and phage vector molecules. lambda pMYF11 is a hybrid of lambda 47.1 vector and pBR322 plasmid. CI- marker of pMYF11 is replaced with cI+ marker by recombination between the plasmid and prophage 434. The phasmid molecule can be used as a replacement vector for BamHI, HindIII, SalGI endonucleases. The maximum size of fragments to be cloned is 21 kilobase pairs. Positive selection for hybrid molecules is possible because of the Spi phenotype expression after replacement of the central HindIII or BamHI DNA fragment with foreign DNA. A library of Escherichia coli genes is constructed with the help of lambda pMYF11 as a vector molecule. A hybrid phage harboring genes of the proline operon is detected by means of complementation.     

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.