Restriction endonuclease mapping and cloning of Mycobacterium intracellulare plasmid pLR7

A restriction map of Mycobacterium intracellulare plasmid pLR7 was developed. This 15.3-kb plasmid had unique sites for BamHI, HindIII, and XbaI. Various large fragments of pLR7 were cloned into pBR322 or pHP34 and propagated in Escherichia coli. A hybrid pLR7 ::pBR322 plasmid carrying the complete pLR7 sequence was constructed by joining the plasmids at their HindIII sites. The construction of these hybrids will facilitate the analysis and manipulation of pLR7 and may allow the development of this plasmid as a model system for genetic analysis in mycobacteria.     

Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene

The DNA sequence of a 1.45 kb EcoRI fragment from the yeast (Saccharomyces cerevisiae) TRP1 region has been determined. The fragment contains the TRP1 gene and a yeast chromosomal replicator. The TRP1 gene has been located on the fragment by analysis of potential initiation and termination codons in the DNA sequence. This location has been confirmed by subcloning portions of the fragment. Both the 5' and 3' noncoding regions of the TRP1 gene contain sequence homologies with analogous areas surrounding other yeast genes. The yeast replicator has been localized in a region near the 3' end of the TRP1 gene. The DNA sequence in this region contains several structural features which may be involved in the initiation of DNA replication.     

The nucleotide sequence of IS5 from Escherichia coli

A 3-kb fragment of Haemophilus haemolyticus DNA which carries the HhaII restriction (r) and modification (m) genes has been cloned into the PstI site of pBR322 (Mann et al., 1978). When propagated in Escherichia coli, it was observed that spontaneous insertions of IS5 inactivated the restriction gene, producing r- mutants at a frequency of 10(-6). Electron microscopy, restriction-site mapping and sequence analysis of two r- plasmids have demonstrated the presence of IS5 at a single target site in both possible orientations. The complete nucleotide sequence of IS5 has been determined. It is 1195 bp long and has inverted terminal repeats of 16 bp. The target site for IS5 in this plasmid is 5'-CTAG. Approx. ten copies of IS5 were found to be present at about the same locations on the E. coli chromosome in various K-12 strains, using Southern hybridization analysis.     

Mitochondrial DNA from lupine: restriction analysis and cloning of fragments coding for tRNA

Mitochondrial DNA (mtDNA) was isolated from lupine. Restriction analysis was used to estimate its size, which is about 180 kb. A BamHI bank of this mtDNA was constructed using plasmids pBR322 and pBR327 as vectors. Eight clones containing plasmids hybridizing to mitochondrial tRNA (mttRNA) were isolated. Restriction maps of these plasmids were determined. Six of these plasmids hybridized to unique fragments and two to two fragments of very similar size, all obtained by BamHI cleavage of mtDNA.     

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.     

Cloning of the ColE3-CA38 colicin and immunity genes and identification of a plasmid region which enhances colicin production

The colicin and immunity genes of plasmid ColE3-CA38 have been localized by characterization of bacteria carrying its cloned restriction fragments. They are within a 3.14-kb EcoRI segment, such that the immunity gene contains the KpnI site, and the colicin gene is adjacent to it within a 2.1-kb KpnI-HincII segment. The immunity gene and one end of the colicin gene are in the region of ColE3-CA38 which is not homologous to the closely related plasmid ColE2-P9. A 0.64-kb PvuI-EcoRI segment of the plasmid adjacent to that containing the colicin and immunity genes was found to augment colicin production on solid media, and also affected the morphology of clearing zones produced by the cells when used as indicators in overlays of stabs of colicin E2 or E7 producers. The 0.64-kb segment was required in its native orientation relative to the 3.14-kb EcoRI segment to cause its effects.     

Cloning of the vaccinia virus telomere in a yeast plasmid vector

The vaccinia virus DNA telomere, which contains a covalently closed hairpin structure, has been cloned in a yeast plasmid vector. Restriction mapping indicates that the cloned vaccinia telomere is maintained in yeast not in its native hairpin configuration but as an inverted repeat structure, within a circular plasmid, with the sequences of the viral hairpin now at the axis of symmetry of an imperfect palindrome. As such, the cloned telomere resembles the telomeric replicative intermediate observed during vaccinia virus DNA replication. Small deletions and duplications in the viral inverted repeats of different clones suggest a model in which the observed circular plasmids were generated in yeast by the replication of hybrid linear DNA molecules consisting of the linearized yeast vector flanked by two hairpin-containing vaccinia termini.     

Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha amylase

A resident-plasmid cloning system developed for Bacillus subtilis has been used to isolate recombinant plasmids carrying DNA from Bacillus licheniformis which confer alpha-amylase activity on alpha-amylase-negative mutants of B. subtilis. These plasmids contain a 3550-bp insert at the EcoRI site of the plasmid pBD64. Subcloning various lengths of the B. licheniformis DNA has localised the gene to a 2550-bp BclI fragment. We present evidence that the cloned fragment codes for a B. licheniformis heat-stable alpha-amylase with a temperature optimum of 93 degrees C. The foreign gene is expressed efficiently in B. subtilis and is stably maintained.     

In vitro and in vivo manipulations of bacteriophage Mu DNA: cloning of Mu ends and construction of mini-Mu's carrying selectable markers

Recombinant plasmids carrying one or both ends of the bacteriophage Mu genome were constructed by molecular cloning. Transposable mini-Mu's with selectable markers (ampicillin resistance, kanamycin resistance or the entire lac operon of Escherichia coli) inserted between the Mu ends were also constructed. As a source of lac operon DNA, a pBR322 derivative with a 27 kb insert containing the lac operon was constructed. The plasmids with both ends of Mu (mini-Mu's) conferred full Mu immunity upon the host cells. However, the same mini-Mu's containing kan or lac inserts were defective in immunity. A summary of the construction and physical characterization, including restriction endonuclease cleavage maps and some of the biological properties of the plasmids, is presented.     

The ori sequences of the mitochondrial genome of a wild-type yeast strain: number,location, orientation and structure

We have investigated the number, the location, the orientation and the structure of the seven ori sequences present in the mitochondrial genome of a wild-type strain, A, of Saccharomyces cerevisiae. These homologous sequences are formed by three G + C-rich clusters. A, B and C, and by four A + T-rich stretches. Two of the latter, p and s, are located between clusters A and B; one, l between clusters B and C; and one r, either immediately follows cluster C (in ori 3–7), or is separated from it by an additional A + T-rich stretch, r', (in ori 1 and ori 2). The most remarkable differences among ori sequences concern the presence of two additional G + C-rich clusters,β and γ, which are inserted in sequence l of ori 4 and 6 and in the middle of sequence r of ori 4,6 and 7, respectively. Neglecting clusters /gb and γ and stretch r', the length of on sequences is 280 ± 1 bp, and that of the l stretch 200 ± 1 bp. Hairpin structures can be formed by the whole A-B region, by clusters β and γ, and (in ori 2–6) by a short AT sequence, lp, immediately preceding cluster /gb. An overall tertiary folding of ori sequences can be obtained. Some structural features of ori sequences are shared by the origins of replication of the heavy strands of the mitochondrial genomes of mammalian cells.     

A simple method for shortening a plasmid genome using a system of plasmid cointegration mediated by a Tn3 mutant

This paper describes a simple method for the isolation of small plasmids of various sizes from pSMI, a derivative of the resistance plasmid R 100. The method is based on the observation that a repressor-negative mutant of the ampicillin-resistance (amp^r) transposon Tn3, Tn3 No. 5, mediates cointegration of a plasmid carrying Tn 3 No. 5 (pMB8::Tn 3 No. 5) into virtually any site on pSMI. The resulting cointegrate plasmids contain the pSMI sequence which is joined with the amp^r gene of the Tn 3 mutant. This cointegration is so frequent that large cointegrate plasmids can be readily detected in the total plasmid DNA prepared from cells carrying pSMI and pMB8::Tn3 No. 5. We were able to isolate small plasmids of various sizes by digesting the total plasmid DNAs with restriction endonucleases which cut both pSM 1 and Tn3 No. 5 sequences present in the cointegrates and subsequently ligating the restriction fragment containing both the amp^r gene and the region necessary for replication of pSMI. Analysis of these plasmids, named pBV plasmids, with restriction endonucleases and by nucleotide sequencing allowed us to determine regions necessary or unnecessary for replication, thus defining a minimal replication region of pSMI. The present method is generally useful for the isolation of small derivatives from any large plasmid for the study of genes and sites adjacent to or within the minimal replication region of the plasmid.     

Cloning of a gene required for tryptophan biosynthesis from Leptospira biflexa serovar patoc into Escherichia coli

A clone bank, consisting of approx. 8100 colonies, has been created for the spirochete Leptospira biflexa serovar patoc in Escherichia coli using pBR322 as the vector. One of these clones contains the genetic information needed to complement a defect in the trpE gene of E. coli. The information resides on a 20.5-kb plasmid designated pYCl, which carries a 16-kb insert consisting of three HindIII fragments. It does not complement defects in other genes needed for the biosynthesis of tryptophan in E. coli.     

Lethality of palindromic DNA and its use in selection of recombinant plasmids

A plasmid derived from ColE1 is constructed so that the removal of one restriction endonuclease HindIII fragment allows the ends of the remaining single fragment (the replicator) to be joined, generating a palindromic sequence 2394 bp in length. The circular species thus produced gives rise to transformants of E. coli at very low frequency. Since the palindromic sequence is effectively lethal to a plasmid containing it, the replicator will give rise to more transformants when the restriction fragment originally removed from it is replaced by another. This principle can be exploited to allow the efficient molecular cloning of unselected restriction fragments.     

Purification of a new restriction endonuclease, StyI, from Escherichia coli carrying the hsd+ miniplasmid

A new restriction endonuclease, StyI, free of contaminating nuclease activities, has been isolated from Escherichia coli carrying the hsd+ miniplasmid of Salmonella typhi origin. In the presence of 10 mM Mg2+, it recognizes and cleaves a hexanucleotide sequence of 5'-C decreases C(AT)(AT)GG. The advantages of the StyI endonuclease include its stability, high yield (more than 2 X 10(3) units/g of wet cells), easy handling of producer cells, and the ability to recognize new sequences, CCAAGG and CCTTGG.     

Recombination between satellite phage P4 and its helper P2. I. In vivo and in vitro construction of P4: :P2 hybrid satellite phage

P4::P2 hybrid satellite phages which carry a portion (including the P2 head gene Q and the cohesive end) of the left end of the P2 chromosome linked to the essential part of the P4 chromosome have been isolated by in vivo as well as in vitro recombination. These hybrids express gene Q and grow in the presence of a P2 helper even if defective in gene Q.     

An unstable mutant gene of the am locus of Neurospora results from a small duplication

We have cloned the unstable am mutant gene, am126, as well as the am gene from an am126 revertant. The mutation is a result of a 33-bp duplication that repeats a sequence starting 13 bp upstream of the 3' splice junction between intron 1 and exon 2 and extends 20 bp into exon 2. In addition, there is a G----A transition 2 bp upstream of the first copy of the duplicated sequence. In the revertant gene the wild-type sequence is precisely recovered, involving both the loss of the duplication and a reversion (A----G) of the associated transition. Our data suggest that only the more 5' of the two 3' splice junctions present in the duplicated version of the gene is used. This favors a 5'----3' scanning mechanism for exon splicing.     

Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli

The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity.     

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.