Internal Restriction Sites Research Articles

A simplified procedure for cDNA and genomic library construction using nonpalindromic oligonucleotide adaptors

The properties and characteristics of oligonucleotide adaptors for use in a simplified procedure for the construction of cDNA and genomic DNA libraries are described. The adaptors are suitable for joining to blunt ended cDNA or sheared genomic DNA, and then to the cohesive ends of restriction sites in vectors. Each adaptor consists of two oligonucleotides with complementary but nonpalindromic sequences that include an internal restriction site, a 5' phosphorylated blunt end, and an overlapping or staggered 5' hydroxylated end corresponding to a restriction endonuclease site in a vector of choice. Ligation of the blunt end to high molecular weight target DNA proceeds efficiently and there is no tandem concatenation of the adaptor. Insertion into the appropriate vector only requires ligation of the cohesive ends. There is no requirement for methylation, restriction enzyme cleavage, G-C tailing, or denaturation after ligation of the adaptor to the target DNA, all characteristics of other procedures.

Transposable element‐induced mutations of the viviparous‐1 gene in maize

AbstractThe viviparous‐1 (vp1) locus in maize is a developmental gene that controls diverse aspects of the maturation phase of seed development. Mutations of vp1 alter embryo sensitivity to the hormone abscisic acid and block formation of anthocyanin pigment. Molecular cloning of a Robertson Mutator‐induced mutant allele, vp1‐mum‐1, by transposable element tagging has allowed analysis of several transposon‐induced vp1 mutants. In the vp1‐Mc mutation, the gene is disrupted by 4.0 kbp insertion, which results in expression of a 3′ truncated mRNA. Phenotypically, this allele is at least partially functional in causing embryo dormancy, but is ineffective in controlling anthocyanin expression. This result suggests that disruption of the C‐terminal domain of the Vp1 protein specifically affects regulation of the anthocyanin pathway. A second Mutator‐ derived allele, vp1‐mum2, exhibits an unusual form of somatic mutability in which endosperm cells revert from wild‐type vp1 expression to a mutant condition. The vp1‐mum2 allele contains a 1.5 kbp Insertion that has no detectable homology to known Mu elements. This element is retained In wild‐type germinal revertants derived from vp1‐mum2 An apparent DNA modification affecting cleavage at an internal Sstl restriction site in the element correlates with vp1‐mum2 states that exhibit wild‐type Vp1 expression. A model involving mitotic assortment of modified and unmodified DNA strands during development is proposed for vp1‐mum2 somatic mutation.

Expression of herpes simplex virus type 1 DNA polymerase gene by in vitro translation and effects of gene deletions on activity.

A cloned herpes simplex virus type 1 DNA polymerase gene which is biologically functional was inserted into SP6 and T7 promoter-containing vectors for in vitro transcription-translation. pol-specific RNA synthesized in vitro will direct the synthesis of a 140-kilodalton polypeptide in rabbit reticulocyte lysates. RNAs prepared from pol templates linearized at internal restriction sites specified deleted polypeptides with sizes consistent with colinearity of the pol gene and the 140-kilodalton primary translation product. The in vitro translated pol gene product was enzymatically active, with salt resistance and sensitivity to acyclovir triphosphate, similar to the enzyme activity in crude extracts of herpes simplex virus type 1-infected Vero cells. An in-frame deletion of 78 residues (amino acids residues 881 to 959) was introduced into the expression vectors to investigate the function of a region of the polypeptide (amino acids residues 881 to 895) which is conserved in nine other DNA polymerases. In a complementation assay, this mutation abolished biological activity as well as the enzymatic activity of the in vitro translated product. A BAL31 mutation deleting the upstream open reading frame of pol had no effect on biological activity in a complementation assay but was found to increase the efficiency of in vitro translation of pol RNA. Two amino-terminal deletions of 27 and 67 residues were found to greatly enhance the enzymatic activity of the in vitro translated product, while all carboxy-terminal deletions examined (the smallest being 164 residues) abolished in vitro enzymatic activity. Expression of the 67-residue amino-terminal deleted pol gene in Escherichia coli, using a bacteriophage T7-based system, resulted in accumulation of large amounts of an insoluble fusion protein. An antiserum prepared against this fusion protein precipitated the 140-kilodalton DNA polymerase from herpes simplex virus type 1-infected cell lysates.

Total chemical synthesis of a gene for hepatitis B virus core protein and its functional characterization

We have chemically synthesized a DNA duplex of 560 nucleotides that codes for the hepatitis B virus (HBV) core protein. The synthetic gene contains 27 unique internal restriction sites. Thereby, it can easily be mutagenized by replacement of rather short restriction fragments. A number of restriction recognition sequences are in common between the synthetic and the authentic gene, thus allowing for the transfer of synthetic segments into the cloned viral genome. Several unexpected mutations in the synthetic gene were readily corrected utilizing the multiple unique restriction sites. In Escherichia coli, the expression level of the synthetic gene product amounts to about 4% of the total soluble protein. It forms particles closely resembling native HBV cores. After transfer of the synthetic gene into the viral genome, transient expression in a hepatoma cell line yields proteins indistinguishable from the native gene products. The synthetic gene thus provides a useful tool for studies on the structure and function of the isolated HBV core protein as well as the gene and its various products in the viral life-cycle.

Distribution of insertion sequence ISRm1 in Rhizobium meliloti and other gram-negative bacteria.

An internal 0.9 kb segment of Rhizobium meliloti insertion sequence ISRm1 was used as a probe to determine the distribution of ISRm1 in strains of R. meliloti and other Gram-negative bacteria. The insertion sequence was detected in 80% (12/15) of R. meliloti strains from different parts of the world. Its copy number ranged from one to at least eleven. The ISRm1 copies detected showed variation in their internal restriction sites and their degree of homology to the probe. ISRm1 was found in a variety of genomic restriction fragments, and was detected in plasmids, including the nod and exo megaplasmids of R. meliloti. Other rhizobia found to contain ISRm1 were a strain of R. leguminosarum biovar phaseoli and two Rhizobium isolates capable of nodulating both Medicago sativa and Phaseolus vulgaris. It was also found in a diazotrophic soil bacterium isolated from the roots of wetland rice.

Cloning of the carbohydrate-binding portion of the toxin A gene ofClostridium difficile

A portion of the toxin A gene ofClostridium difficile was cloned into pBR322 withEscherichia coli Chi 1776 as the host. Five identical clones, each containing a 4.7-kbPstI restriction endonuclease fragment and producing toxin A antigens, were detected with affinity-purified, monospecific antibodies against toxin A. Digestion of the cloned DNA withPstI revealed as internal restriction site that resulted in two fragments (2.1 and 2.6 kb in size). Probe DNA from either of these fragments hybridized with DNA in the 4.7 kb region ofPstI-digested, high-molecular-weight DNA from the sourceC. difficile strain, indicating that the internalPstI site is protected. The probe DNA also hybridized with restriction-digested DNA from five additional toxigenic strains, but it did not hybridize with DNA from four nontoxigenic strains. In addition, a DNA fragment from a toxigenic strain ofClostridium sordellii, whose toxin cross-reacts with antibody toC. difficile toxin A, hybridized with the clonedC. difficile DNA. Unlike native toxin A, the cell lysate from the recombinant clone was not cytotoxic to Chinese hamster ovary cells or enterotoxic in hamsters. It did agglutinate rabbit red blood cells, a characteristic of toxin A. The cell lysate also exhibited a line of partial identity when compared with purified toxin A in Ouchterlony assays, and it reacted with monoclonal antibody to toxin A in an enzyme-linked immunosorbent assay. The cloned DNA appears to code for a nontoxic binding portion of toxin A, which is responsible for binding to galactose-α1-3-galactose-β1-4-N-acetylglucosamine.

Fertility inhibition gene of plasmid R100.

The fin0 gene of R100 was isolated from the Fin0+ transducing phage VA lambda 57. The limits of the gene were determined by BAL31 digestions and by analysis of deletion mutations derived from an internal restriction site. The DNA sequence contained an open reading frame of 558 nucleotides that would encode a protein of 21,268 daltons. Synthesis of such a protein was observed only when the fragment was cloned in front of the TAC promoter. Deletions entering the large open reading frame from either end were Fin0-, while internal frame shift mutations retained high Fin0 activity. One such strain had a 13 bp internal deletion that would produce a protein of 63 amino acid residues of which 21 were basic. We were consequently unable to rigorously establish that the 558 base orf encoded a fin0 product. The strand opposite the large open reading frame contained several transcription termination signals, and it is possible that the active gene product is one or two small RNAs from this strand.

Sex-dependent expression of mouse testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha): cDNA cloning and pretranslational regulation.

By using both double-colony hybridization and an in situ immunostaining assay for transformants, 39 cDNA clones (clone p-16 alpha) encoding mouse liver microsomal testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha) were isolated from a cDNA library constructed in the cloning vector pUC-9 with poly(A)+ RNA immunoenriched from total liver polysomes of male 129/J mice. mRNA selected by hybridization with clone p-16 alpha translated the P-450(16) alpha apoprotein in vitro. Total cellular proteins, which were prepared from immunopositive transformant Escherichia coli cells, were conjugated with Sepharose 4B. Antibody purified with the Sepharose 4B conjugate from mixed antiserum to P-450(16) alpha and P-450(15) alpha specifically inhibited testosterone 16 alpha-hydroxylase activity in microsomes. The cDNA insert of one recombinant plasmid (clone P-16 alpha-1) was 1.75 kilobases in size and contained one or more internal restriction sites for HindIII, BamHI, Bgl I, Pst I, Alu I, HinpI, and Rsa I. 32P-labeled clone p-16 alpha-1 hybridized with a single mRNA (2000 bases) that was 10 times more concentrated in liver cells from male 129/J mice than in female mice. This result was consistent with the finding that poly(A)+ RNA from male mice translated 10 times as much P-450(16) alpha in vitro as did the poly(A)+ RNA from females. Thus, the predominant expression of testosterone 16 alpha-hydroxylase in male 129/J mice is regulated pretranslationally, presumably at the transcriptional level of the P-450(16) alpha gene.

Screening of M13 mp bacteriophage clones for small inserts by primer extension and insert excision

The M13 bacteriophage was adapted by Messing for DNA cloning by insertion of sequences containing a β galactosidase gene with internal restriction sites. Cloning into these sites results in loss of β galactosidase activity, allowing for initial screening of plaques for insert DNA but sometimes producing false positives. In our hands, previously described methods for the further screening of phage for short inserts have limitations that are overcome by the method described here. In our method, a commercially available primer is annealed to single-stranded phage DNA at a sequence that lies immediately 5′ to the cloning site within the β galactosidase gene. Klenow fragment and 4 deoxynucleotide triphosphates are then employed to extend the primer, forming double-stranded DNA, which is cleaved at restriction sites flanking the cloning site. Agarose gel electrophoresis with ethidium bromide staining is used to separate phage from insert DNA, thus demonstrating the presence of cloned insert.

Characterization and chromosomal location of endogenous mouse mammary tumor virus loci in GR, NFS, and DBA mice

Endogenous mouse mammary tumor virus (MMTV) proviral copies were characterized in three genetically dissimilar mouse strains: GR, a high-tumor-incidence strain bred in Europe that carries an MMTV proviral copy associated with early mammary tumors; DBA, a high-tumor-incidence laboratory strain bred in the USA with an endogenous copy that is associated with MMTV antigen expression in the milk; and NFS, a recently inbred line of the low-tumor-incidence NIH Swiss mouse. MMTV proviral loci were studied using restriction endonuclease analysis and the Southern transfer procedure in genetic crosses and in somatic cell hybrids. By studying the segregation of MMTV-specific EcoRI, BamHI, and PstI fragments, the organization of these fragments into MMTV proviral loci was determined and it was shown that (1) many homologous proviral loci are present in these three mouse strains, (2) these MMTV proviruses differ in their pattern of internal restriction sites, and (3) the MMTV loci are distributed on multiple chromosomes including 1 and 7.

Double cos site vectors: simplified cosmid cloning

A new vector for construction of cosmid libraries is described. Cosmid c2XB contains restriction sites for use in the insertion of foreign DNA and two λ cos sites separated by a blunt-end restriction site. The presence of two cos sites on a single plasmid eliminates the need to prepare two separate cosmid arms, and the internal blunt-end restriction site prevents cosmid concatemerization. Thus, a double restriction-enzyme digestion is sufficient to prepare the vector for subsequent ligation with DNA fragments which are dephosphorylated to prevent their self-ligation. The use of this vector system allows efficient cosmid cloning (1 × 10 5 colonies per μg insert DNA) and eliminates background due to vector self-ligation. Furthermore, the procedure is so rapid as to eliminate the need to amplify cosmid libraries for storage and reuse. Also described is a cosmid vector for use in construction of cosmid libraries which are to be introduced into cultured eukaryotic cells. This vector contains the Herpex simplex virus thymidine kinase (HSV tk) gene as a selectable marker and a retroviral long terminal repeat (LTR) region as an enhancer sequence.

Localization of genes responsible for replication and immunity to colicin A on plasmid ColA-CA31.

The region containing the origin and regulatory sites for replication as well as the immunity gene (iaa) have been localized on the plasmid ColA-CA31. The region involved in replication functions of ColA can be hybridized with that of ColE1. It is located between 1 and 1 kb on the plasmid map previously published (Morlon et al. 1982a). A 0.50 Kb HincII fragment of ColA can be weakly hybridized to the ColE1 immunity region. This fragment contains iaa since directed in vitro mutagenesis at an internal restriction site can abolish the immunity to colicin A; however, it does not contain the entire iaa. Knowing the localization of regions involved in autonomous DNA replication and immunity, a mini-ColA plasmid was constructed that contains these two regions. The mini-ColA of 2.8 Kb can be amplified in the presence of chloramphenicol and confers the immunity to transformants. It thus constitutes a useful cloning vector. Expression of ColA and of the various constructed plasmids in the maxicell system suggests that the immunity protein has a molecular weight of about 18-20 Kd.

Isolation of a cDNA clone for human antithrombin III.

Antithrombin III (ATIII) is an important plasma protease inhibitor with a central role in the coagulation system. On the basis of its protein sequence, ATIII is one member of a "super family" of protease inhibitors that includes alpha 1-antitrypsin and chicken ovalbumin. An increased risk of thromboembolism is associated with inherited ATIII deficiency. To study the structure and expression of the human ATIII gene, we have isolated complementary (cDNA) clones for ATIII from human liver mRNA. ATIII cDNA clones were identified by hybridization to a mixture of synthetic oligodeoxynucleotides encoding amino acids 251-256 of the ATIII protein sequence. The largest cDNA clone (1.4 kilobases) included the coding region of ATIII mRNA from codon 10 through a 3'-untranslated region. Comparison of ATIII cDNA clones from two different sources revealed a sequence polymorphism at an internal PstI restriction site. Analysis of both total genomic DNAs and an ATIII gene cloned in a bacteriophage Charon 4A showed that the ATIII gene is present once per haploid genome and is distributed over 10-16 kilobases of DNA. Computer-assisted comparison of the cDNA sequence with those for baboon alpha 1-antitrypsin and chicken ovalbumin revealed homologies consistent with their inclusion in the protease inhibitor superfamily.

Zein storage protein gene family of Maize: An assessment of heterogeneity with cloned messenger RNA sequences

Zein messenger RNAs are shown to have an average poly(A) tract of 110 residues and a G + C content of 50%. Duplex DNA was enzymatically synthesized using purified zein m RNAs as the template material and inserted into a bacterial plasmid vector. Clones were rapidly screened for the size of the DNA insert by direct extraction of DNA from single colonies. A set of 20 clones with varying sizes was selected for further characterization. The 18 recombinant clones that contained zein sequences hybridized specifically to mRNA encoding polypeptides in either the light or the heavy zein class. The clones were further subgrouped by the presence of similar internal restriction sites and by the relative degree of sequence homology. The clones could be divided into two heavy class subgroups and at least three light class subgroups. In hybridizations of Southern transfers, representative clones from each of four subgroups reacted with numerous restriction fragments of genomic DNA. While each probe appeared to recognize common genomic sequences at the stringency employed, they each also hybridized to other bands. These results substantiate the contention that zein is a multigene family composed of sequences with limited heterogeneity. Although zein is being synthesized in large amounts in the endosperm at mid-development, no rearrangements or amplification of genomic endosperm sequences seem to be implicated.