Mary C. Betlach

Construction and characterization of new cloning vehicles. II. A multipurpose cloning system

In vitro recombination techniques were used to construct a new cloning vehicle, pBR322. This plasmid, derived from pBR313, is a relaxed replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc). The antibiotic-resistant genes on pBR322 are not transposable. The vector pBR322 was constructed in order to have a plasmid with a single PstI site, located in the ampicillin-resistant gene (Apr), in addition to four unique restriction sites, EcoRI, HindIII, BamHI and SalI. Survival of Escherichia coli strain X1776 containing pBR313 and pBR322 as a function of thymine and diaminopimelic acid (DAP) starvation and sensitivity to bile salts was found to be equivalent to the non-plasmid containing strain. Conjugal transfer of these plasmids in bi- and triparental matings were significantly reduced or undetectable relative to the plasmid ColE1.

Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9

In vitro recombination via restriction endonucleases and the in vivo genetic translocation of the Ap resistance (Apr) gene resulted in the construction of a new cloning vehicle, the plasmid pBR313. This vector was derived from a ColE1-like plasmid and, while it does not produce colicon E1, it still retains colicin E1 immunity. The Apr and tetracycline resistance (Tcr) markers carried in pBR313 were derived from the ampicillin transposon (TnA) of pRSF2124 and pSC101 respectively. During the construction of pBR313, the TnA component was altered and the Apr gene in pBR313 can no longer be translocated. This plasmid has a molecular weight of 5.8 Mdalton and has been characterized using thirteen restriction enzymes, six of which (EcoRI, SmaI, HpaI, HindIII, BamHI and SalI) cleave the plasmid at unique restriction sites. This allows the molecular cloning of DNA fragments generated by these six enzymes. The restriction sites for the latter three enzymes, HindIII, BamHI and SalI, are located in the Tcr gene(s). Cloning DNA fragments into these sites alters the expression of the Tcr mechanisms thus providing a selection for cells carrying recombinant plasmid molecules. An enrichment method for AprTcS cells carrying recombinant plasmid molecules is described.

Protein expression in E. coli minicells by recombinant plasmids

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.

CONSTRUCTION AND CHARACTERIZATION OF CLONING VEHICLES

ABSTRACT A series of in vitro recombination experiments with restriction endonuclease fragments of pSClOl and a col El-like plasmid resulted in the recovery of a plasmid containing the best cloning characteristics of each plasmid. The newly constructed plasmid, designated pMB9, is similar to col El in its replication properites, its immunity to colicin E1 but does not produce colicin E1, and does not appear to form a relaxation complex. It carries a segment of DNA derived from the pSClOl plasmid which contains a functional tetracycline resistant gene. The plasmid has one restriction site for the Eco RI, Hind III, Bam I and Sal I restriction endonucleases. The latter three sites are located in the gene for tetracycline resistance. The plasmid has a molecular weight of about 3.5 × 10 6 daltons. A derivative of pMB9 containing an ampicillin resistant gene was constructed by in vivo translocation of this gene from p2124 to pMB9. This new plasmid, designated pBR312, contains two BamI sites and was used as a parent for the construction of several plasmids which can serve as cloning vehicles for Hind III, Sal I and Bam I endonuclease generated fragments by providing ampicillin resistance as a selective marker. Transformants containing recombinant plasmids with DNA fragments inserted at any of these three sites can be identified or selected from transformant populations by their response to tetracycline.

Genome organization in Halobacterium halobium: a 70 kb island of more (AT) rich DNA in the chromosome.

SummaryThe more A+T rich fractionated component (FII DNA) of the Halobacterium halobium genome constitutes one third of the total DNA and upon isolation consists of covalently closed circular DNA (pHH1 and minor cccDNA) and nonsupercoiled sequences. We have investigated the physical organization of the non cccDNA in FII by a chromosome walk using one copy of the halobacterial insertion element ISH1 as a start point. This chromosome walk led to the isolation of 160 kb of chromosomal DNA containing 70 kb of FII DNA covalently linked to more G+C rich sequences (FI DNA). Copies of three previously characterized insertion elements (ISH1, ISH2, and ISH26) as well as at least 10 other repeated sequences are clustered within this chromosomal FII DNA “island”. Unique sequences are found in the FI DNA flanking the FII DNA island as well as in 40 kb of FI DNA surrounding the bacterio-opsin gene. The presence of pHH1 in H. halobium and closely related species correlates with the occurrence of the characterized chromosomal FII DNA island. Halophilic purple membrane producing isolates YC81819-9, GN101, SB3 and GRA lack pHH1 and the 70 kb FII DNA, but contain all of the FI DNA sequences tested. We propose that pHH1 and this chromosomal FII DNA are characteristic genomic components of H. halobium and closely related species, and, that the 70 kb FII DNA might represent a large insertion in the chromosome of H. halobium and closely related species. The conservation of both FI and FII DNA sequences can be used for strain classification and determination of evolutionary relationships among halobacteria.

Transposable elements of halobacterium-halobium

SummaryFive different DNA insertions (ISH1, ISH2, ISH23, ISH24, and ISH25) are found in or upstream of the bacterio-opsin (bop) gene in Bop mutants of H. halobium. These insertions have been cloned and characterized. They range in size from 520–3,000 bp, and four of the five insertions have structural features similar to known transposable elements. Two of the elements (ISH1 and ISH2) are found in the majority of Bop mutants. The former integrates at a preferred target site, while the latter integrates at numerous sites. The copy number of each insertion element was determined along with its distribution in the 68% G+C (FI), 58% G+C (FII) and cccDNA fractions of the H. halobium genome. There are eight copies of ISH2, one copy in FI and seven in the cccDNA. There are two copies of each of the other three insertion elements and one copy of ISH25. ISH25 does not seem to have the usual structural features of a transposable element. Most of the copies of these insertion elements are located in the cccDNA. Vacuole, ruberin and purple membrane mutants of H. halobium occur at high spontaneous frequencies. Rearrangements, insertions and deletions occur concurrently in the FII and cccDNA of these mutants including insertions of these five elements. Although the bacterio-opsin gene incurs insertions frequently, the 40 kb of flanking FI DNA remains conserved in mutant derivatives of H. halobium and in a number of related species.

Effects of upstream deletions on light‐ and oxygen‐regulated bacterio‐opsin gene expression in Halobacterium halobium

The bacterio‐opsin gene (bop) of Halobacterium halobium is located within a cluster with three other genes. Growth conditions of high light intensity and low oxygen tension induce bop gene cluster expression. To identify putative regulatory factor binding sites upstream of the bop gene, we have compared sequences upstream of the bop gene with the corresponding sequences from two other genes in the bop gene cluster. Conserved sequence motifs were observed which may mediate the effect of high light intensity and/or low oxygen tension on bop gene expression. Based on these motifs, a set of mutants was constructed which contained deletions upstream of the bop gene. These constructs were tested in a host strain where bop gene expression is independent of oxygen regulation and in another strain where it is regulated by oxygen and light. The minimal upstream sequence required for both light‐ and oxygen‐regulated bop gene expression was determined to be 54 bp.

Two-dimensional crystallization of Escherichia coli-expressed bacteriorhodopsin and its D96N variant: high resolution structural studies in projection.

Highly ordered two-dimensional (2-D) crystals of Escherichia coli-expressed bacteriorhodopsin analog (e-bR) and its D96N variant (e-D96N) reconstituted in Halobacterium halobium lipids have been obtained by starting with the opsin protein purified in the denaturing detergent sodium dodecyl sulfate. These crystals embedded in glucose show electron diffraction in projection to better than 3.0 A at room temperature. This is the first instance that expressed bR or a variant has been crystallized in 2-D arrays showing such high order. The crystal lattice is homologous to that in wild-type bR (w-bR) in purple membranes (PM) and permit high resolution analyses of the structure of the functionally impaired D96N variant. The e-bR crystal is isomorphous to that in PM with an overall averaged fractional change of 12.7% (26-3.6-A resolution) in the projection structure factors. The projection difference Fourier map e-bR-PM at 3.6-A resolution indicates small conformational changes equivalent to movement of approximately < 7 C-atoms distributed within and in the neighborhood of the protein envelope. This result shows that relative to w-bR there are no global structural rearrangements in e-bR at this 3.6 A resolution level. The e-D96N crystal is isomorphous to the e-bR crystal with a smaller (9.2%) overall averaged fractional change in the structure factors. The significant structural differences between e-D96N and e-bR are concentrated at high resolution (5-3.6 A); however, these changes are small as quantified from the 3.6 A resolution e-D96N-e-bR Fourier difference map. The difference map showed no statistically significant peaks or valleys within 5 A in projection from the site of D96 substitution on helix C. Elsewhere within the protein envelope the integrated measure of peaks or valleys was < approximately 3 C-atom equivalents. Thus, our results show that for the isosteric substitution of Asp96 by Asn, the molecular conformation of bR in its ground state is essentially unaltered. Therefore, the known effect of D96N on the slowed M412 decay is not due to ground-state structural perturbations.

CHARACTERIZATION OF TETRACYCLINE AND AMPICILLIN RESISTANT PLASMID CLONING VEHICLES

Abstract: Our laboratory has been constructing plasmids with suitable genetic properties for the cloning of DNA fragments. We have combined the essential replication properties of the plasmid ColEl with two antibiotic resistance markers (tetracycline and ampicillin) to generate a series of related plasmids. During the course of the construction of these plasmids we have determined the positions of restriction sites used for cloning DNA fragments relative to the antibiotic resistance genes and the origin of DNA replication. We have mapped a number of restriction sites on a series of related plasmids. We will summarize the development of two plasmids, pBR313 and pBR322, which are useful cloning vehicles for in vitro recombinant DNA research. The characterization of these plasmids has prompted an investigation of the nature of tetracycline resistance determined by the plasmid pSC101 and the pMB9 series of plasmids.

RESONANCE RAMAN SPECTRA OF BACTERIORHODOPSIN MUTANTS WITH SUBSTITUTIONS AT ASP‐85, ASP‐96, AND ARG‐82

Detergent solubilized bacteriorhodopsin (BR) proteins which contain alterations made by site‐directed mutagenesis (Asp‐96→Asn, D96N; Asp‐85→Asn, D85N; and Arg‐82→Gln, R82Q) have been studied with resonance Raman spectroscopy. Raman spectra of the light‐adapted (BRLA) and M species in D96N are identical to those of native BR, indicating that this residue is not located near the chromophore. The BRLA states of D85N and especially R82Q contain more of the 13‐cis, C=N syn (BR555) species under ambient illumination compared to solubilized native BR. Replacement of Asp‐85 with Asn causes a 25 nm red‐shift of the absorption maximum and a frequency decrease in both the ethylenic (‐7 cm−1) and the Schiff base C=NH+ (‐3 cm−1) stretching modes of BRLA. These changes indicate that Asp‐85 is located close to the protonated retinal Schiff base. The BRLA spectrum of R82Q exhibits a slight perturbation of the C=NH+ band, but its M spectrum is unperturbed. The Raman spectra and the absorption properties of D85N and R82Q suggest that the protein counterion environment involves the residues Asp‐85−, Arg‐82+ and presumably Asp‐212−. These data are consistent with a model where the strength of the protein‐chromophore interaction and hence the absorption maximum depends on the overall charge of the Schiff base counterion environment.