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The entire mitochondrial (mt) genome of the yeast Schizosaccharomyces pombe (S. pombe) was cloned in the BamHI site of the Escherichia coli plasmid pBR322. Three lines of evidence demonstrate that the complete mtDNA molecule was amplified without rearrangement or partial loss. First, restriction of the hybrid plasmid with BamHI led to the recovery of two fragments corresponding to the linearized plasmid and the BamHI-cut mtDNA. Second, restriction of cloned and native mtDNA with HindIII revealed identical fragments. Third, mitochondrial ribosomal RNA hybridized to the same HindIII fragments from cloned mtDNA and from mtDNA isolated from mitochondria.

Use of restriction endonucleases BglII, EcoRI, BamHI, and HindIII, has established that in small-headed (PIS) virions of coliphage P1, as a population, the entire genome found in big-headed (P1B) virions is represented. In addition, the origin and direction of DNA packaging are identical in P1S and P1B virions.

This paper reports a cleavage site map of Tn5 for restriction enzymes BamHI, Bg/I, Bg/II, Hind II, HindIII, HpaI, Sa/I, Aval, SmaI, XhoI, PstI, PvuII, HaeII and HaeIII that was determined by the analysis of restriction enzyme cleavage patterns of ColEl, two independent ColEl::Tn5 plasmids, and a ColEl::Tn5 deletion derivative. Ba/I, EcoRI, KpnI, and PvuI do not cleave Tn5. Construction and analysis of in vitro-generated deletions of a ColEl::Tn5 plasmid limit the sequences encoding neomycin resistance to a 1500-base-pair-long segment of Tn5. Insertion of DNA at a Bg/II site within this segment results in loss of the neomycin resistance phenotype. Since this Bg/II site lies in an inverted repeat region, sequences within this repeat seem to be involved in the expression of neomycin resistance.

The 2 micron circular DNA from S. cerevisiae has been cloned on bacteriophage lambda. The two forms of circular DNA which exist in equilibrium due to recombination between inverted repeat sequences were separated as stable clones, and a map of targets for restriction endonucleases EcoRI, HindIII and HpaI was constructed. The circular DNAs isolated from a particular oligomycin resistant strain and its parent oligomycin snesitive strain were compared by restriction endonuclease analysis, and no difference was detected. The potential uses of cloned 2 micron DNA in determining the possible biological role of these plasmids are considered.

The conjugative R plasmid R1drd-19, mediating antibiotic resistance to ampicillin (Ap), chloramphenicol (Cm), kanamycin (Km), streptomycin (Sm) and sulfonamides (Su) was mapped using the restriction endonucleases BamHI, HindIII, EcoRI and SalI. BamHI generates 5 fragments (A-E) with molecular weights between 46 x 10(6) 0.25 x 10(6) dalton, and HindIII 8(A-H) between 42 x 10(6) dalton (representing mainly the RTF) and 0.25 x 10(6) dalton (representing the main part of the RTF) and 0.1 x 10(6) dalton. EcoRI recognises 17 sites and produces fragments (A-Q) with molecular weights between 11.7 and 0.1 x 10(6) dalton. SalI yields 7 fragments (A-G) of 16.5 to 2.0 x 10(6) dalton. A physical map was constructed from fragments obtained by partial digestion of R1drd-19 with one restriction enzyme, by double and triple digestion of the DNA with two or three enzymes with and without isolation of individual bands from preparative gels. In addition the restriction patterns of several mutants of R1drd-19 were compared with it. Evidence is presented which indicates that the derivatives of R1 investigated are generated by extended deletions, namely the copy mutant pKN102 which has lost the Km resistance, R1drd-16, which has lost all resistances other than Km and the Kms derivative of R1drd-16, which represents the pure RTF. The map of R1drd-19 is remarkably different from those of R100 and R6-5. Its molecular weight was estimated to be 62.5 Md. The circular fragment order for BamHI is: A-C-B-D-E, for HindIII: A-D-C-B-F-H-E-G, for EcoRI: A-C-K-B-F-J-O-D-H-L-G-P-Q-N-I-E-M- and for SalI A-B-C-D-G-F-E.

The chromosome segment which contains the genes responsible for production of pyocin R2 in P. aeruginosa PAO was defined physically using R-prime plasmids constructed in vivo from R68.45. The previous conclusion from genetic mapping that the cluster of pyocin R2 genes is located in between trpC and trpE genes was confirmed by deletion mapping of various R prime plasmids bearing the trpC gene. The pyocin R2 gene cluster was further localized on two contiguous HindIII fragments of 16 kb and 8.0 kb. PML14 strain, in which R-type pyocin genes were completely deleted, had only one 11 kb HindIII fragment instead. Heteroduplexes between this 11 kb fragment with the two HindIII fragments of PAO revealed that the cluster of pyocin R2 genes was an insertion 13 kb long.

The DNA of bacteriophage T5+ (molecular weight 76 X 10(6) dalton) has been dissected by various specific endonucleases. The restriction enzymes HindIII and EcoRI produce 16 and 7 fragments respectively, whereas Sal and Sma produce 4 fragments each. Complete cleavage maps were established for the enzymes EcoRI, Sal and Sma and an almost complete map for HindIII. Furthermore the location and size of the deletions St 20, St 14, b3, St 0 and b1 were determined. The correlation of the genetic and functional map of the phage with the arrangement of fragments produced by the different enzymes has been established.

The largest HindIII fragment of F includes the entire replication and transfer regions, and its circularisation with ligase gave the conjugative plasmid pED100. This plasmid, which contains none of the F insertion sequences, was essentially unable to mobilise the E. coli chromosome or to give integrative suppression of a dnaA strain.

The streptococcal resistance plasmid pSM10 (8.3 kb), a deletion derivative of pSM10419 (22.9 kb) determining constitutive erythromycin and lincomycin resistance, was physically mapped with the restriction endonucleases AvaI, AvaII, EcoRI, HpaI, KpnI, PvuII (one site each), HindIII, HaeII (three sites each), HincII (four sites), and HhaI (five sites). Using the cryptic plasmid pVA318 as cloning vehicle, the largest HindIII fragment of pSM10 (3.3 kb) was shown to contain the erythromycin/lincomycin resistance gene(s) of the plasmid. The AvaII site of pSM10 proved to be suitable as a site for cloning AvaII-generated chromosomal DNA fragments from a group C streptococcal strain in the Challis strain of Streptococcus sanguis (group) H). A detailed physical map of the chimeric plasmid pSM10221 (12.8 kb), a fusion product of pSM10 and the staphylococcal chloramphenicol resistance plasmid pC221 (4.5 kb), is also presented. The plasmid chimera has properties making it potentially useful in development of a doubly selective streptococcal cloning vehicle by searching for insertional inactivation.

DNA fragments generated by the EcoRI or HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColEl or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants. Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable of supporting replication of a linked ColE1 plasmid in polA− bacteria, were also identified.

We have physically mapped the loci conferring resistance to antibiotics that inhibit mitochondrial protein synthesis (erythromycin, chloramphenicol and paromomycin) or respiration (oligomycin I and II), as well as the 21s and 14s rRNA and tRNA genes on the restriction map of the mitochondrial genome of the yeast Saccharomyces cerevisiae. The mitochondrial genes were localized by hybridization of labeled RNA probes to restriction fragments of grande (strain MH41-7B) mitochondrial DNA (mtDNA) generated by endonucleases EcoRI, HpaI, BamHI, HindIII, SalI, PstI and HhaI. We have derived the HhaI restriction fragment map of MH41-7B mit DNA, to be added to our previously reported maps for the six other endonucleases. The antibiotic resistance loci (antR) were mapped by hybridization of 3H-cRNA transcribed from single marker petite mtDNA's of low kinetic complexity to grande restriction fragments. We have chosen the single Sal I site as the origin of the circular physical map and have positioned the antibiotic loci as follows: C (99.5-1.Ou)--P (27-36.Ou)--OII (58.3-62u--OI (80-84u)--E (94.4-98.4u). The 21s rRNA is localized at 94.4-99.2u, and the 14s rRNA is positioned between 36.2-39.8u. The two rRNA species are separated by 36% of the genome. Total mitochondrial tRNA labeled with 125I hybridized primarily to two regions of the genome, at 99.5-11.5u and 34-44u. A third region of hybridization was occasionally detected at 70--76u, which probably corresponds to seryl and glutamyl tRNA genes, previously located to this region by petite deletion mapping.

1. We have isolated large fragments of the mtDNA of the yeast Saccharomyces carlsbergensis and digested these with restriction endonucleases. The digestion products were separated by electrophoresis in agarose gels. 2. Endonucleases EcoRI, HindII + III, HpaI, HindIII and HapII yield 9, 11, 6, 0 and greater than 80 fragments, respectively. 3. By analysis of partial digestion products and by redigesting the fragments obtained with one endonuclease with a second, we have established the order of all EcoRI and HindII + III fragments. The map is circular and its contour length is 22.1 +/- 0.35 mum, in good agreement with earlier estimates of the size of yeast mtDNA, using electron microscopy and renaturation kinetics. 4. A comparison of the fragmentation pattern of mtDNAs from S. carlsbergensis and various strains of Saccharomyces cerevisiae with endonuclease HindII + III suggests that the overall gene order is similar.

The EcoRI digestion products of phage T4 DNA have been examined using a phage DNA transformation assay. A 2.6 X 10(6) Dalton fragment was found to contain the rII genes. This fragment was purified and then treated with HindIII endonuclease. The cleavage products were ligated to the vector plasmid pBR313 and viable recombinant plasmids recovered. A genetic assay was employed to demonstrate that the recombinants contained T4 DNA and to localize on the phage genetic map the EcoRI and HindIII sites cleaved during the construction of the plasmids. Preliminary characterization suggests that a fragment covering the beginning of the rIIA gene possibly contains a promotor which is active in uninfected cells.

Deletion mutants of bacteriophage Pal 6 were isolated by successive treatments of either heat (60 degrees C) or pyrophosphate (10 mM). These mutants were characterized by restriction enzyme cleavage analysis. The pyrophosphate resistant clones lost the whole Eco R1 fragment in which the Sal I site is located, as well as an unrelated Hind III fragment. These results show that the region containing the Sal I site in the phage genome is not essential for phage viability. This single Sal I site is therefore suitable as a potential insertion site for DNA cloning. On the other hand, the heat resistant clones that were isolated and characterized do not appear to have detectable deletions as indicated by their Eco R1 DNA digestion pattern.

A specialized glpK transducing phage, lambda glpK100, has been isolated and characterized with respect to DNA structure. The glpK component of the glpKF operon has been localized within a 2.0 kilobase pair (kbp) region of the approximately 8.24 kbp bacterial DNA insert, and the positions of BamHI, EcoRI and HindIII restriction sites within this DNA have been identified.

The restriction enzymes BamHI, Bg/II, EcoRI, HindIII, PstI, XbaI and XhoI have been used to cleave DNA isolated from the related coliphages P2 and 186 for analysis on 1% agarose gels. Three approaches were used to map the sites of cleavage: a) analysis dependent upon the existence of cohesive termini and availability of viable P2-186 hybrids; b) analysis of double digests and redigests of isolated fragments with a second enzyme and c) analysis of partial digests by transfer to nitrocellulose and hybridization with a single fragment. This last approach and the results obtained from it are detailed in a separate paper (Saint and Egan, 1979). The number of sites of each enzyme are as follows: a) 186, BamHI-7, Bg/II-1, EcoRI-3, HindIII-2, PstI-22, XbaI-0 and Xho-I-1; b) P2, BamHI-3, Bg/II-2, EcoRI-3, HindIII-0, PstI-O, XbaI-1 and XhoI-O. All of these sites have been mapped with the exception of PstI for 186, where only the five sites in the right 35% (the control region) have been mapped.

Two deletion mutants pAP1 (MW 82 Mdals) and pAP2 (MW 64 Mdals) were isolated by P1 transduction of the plasmid pRD1 (MW 101 (Mdals). These plasmid mutants still carry the his-nif region of K. pneumoniae. They are selftransmissible and mediate resistance to ampicillin, kanamycin and tetracycline. Comparing the HindIII maps of pRD1, pAP1 and pAP2 showed that pAP1 was derived from pRD1 by an 8 μm deletion and pAP2 by two deletions — the same 8 μm deletion and a further 9 μm deletion. The plasmids pAP1 and pAP2 helped us to locate the his-nif region of pRD1 on 3 adjacent HindIII fragments (number 5, 4 and 3 according to gelelectrophoresis). The molecular weights of these fragments were 8.2, 10 and 15 Mdals. These 3 fragments were cloned separately on the multicopy plasmid vehicle pWL625 giving rise to the hybrid plasmids pWK1 (pWL625+HindIII fragment 4), pWK2 (pWL625+HindIII fragment 5). None of these hybrid plasmids conferred nitrogen fixation capacity on E. coli C cells. By combining HindIII fragment 4 and 3 in the same alignment as in pRD1 and cloning them together on pWL625 the hybrid plasmid pWK120 (pLW625+HindIII fragments 4 and 3) was found to carry the entire nif region. An E. coli C strain harbouring the plasmid pWK120 grew on nitrogen free medium and reduced acetylene. The plasmid pWK 120 had a contourlength of 17 μm, a buoyant density of 1.715 g/ml and a copy number up to 65.

The histidine utilization (hut) operons of Klebsiella aerogenes were cloned into pBR322. The hut genes are wholly contained on a 7.9 kilobase pair fragment bounded by HindIII restriction sites and expression of hut is independent of the orientation of the fragment with respect to pBR322. A restriction map locating the 27 cleavage sites within hut for the enzymes, HindIII, PvuII, SalI, BglII, KpnI, PstI, SmaI, AvaI, and BamHI was deduced. Several of the cleavage sites for the enzymes HaeIII and HinfI were also mapped. A set of deletion plasmids was isolated by removing various restriction fragments from the original plasmid. These deletions were characterized and were used to assist in mapping restriction sites. This physical characterization of hut DNA opens the way for genetic and molecular analysis of the regulation of hut gene expression in vitro as well as in vivo.

A restriction endonuclease cleavage map of phage P2 was constructed. The enzymes used and, within parenthesis, the number of their cleavage sites on the P2 lg cc DNA molecule were: AvaI(3), BalI(1), BamI(3), BglII(3), HaeIII (more than 40; only three were mapped), HindIII(0), HpaI(10), KpnI(3), PstI(3), SalI(2) and SmaI(1). The EcoRI cleavage sites (3), as determined earlier, were used as reference points for this study. The DNAs of a variety of P2 mutants carrying chromosomal aberrations (del1, del2, del3, del6, vir22, vir37(2), vir79 and vir94) were also similarly examined.

lambda T4 derivatives that carry T4 tail fiber genes 34-38 have been isolated and characterized by genetic, structural and functional analysis. 32 lambda T4 recombinants were identified by a marker rescue screen of 310 lambda T4 clones generated by restriction of partial cytosine-containing T4 DNA with either HindIII or EcoRI and ligation into appropriately cleaved lambda vectors. These tests defined 15 recombinant classes with respect to the contiguous stretches of genome recovered. Restriction enzyme structural analysis identified 7 HindIII fragments and 7 EcoRI fragments, established a restriction map covering about 11 kb, and indicated the orientation of the DNA inserts within the lambda vectors. The cloned tail fiber genes are expressed efficiently from lambda promoters and complement in vivo T4 phage carrying amber mutations in the tail fiber genes. Polypeptides corresponding to gp34--gp38 have been detected by SDS polyacrylamide gel electrophoresis of 35S-labeled extracts of appropriate lambda T4 recombinant infected UV-treated host cells. The genetic, structural and functional maps of the T4 tail fiber gene cluster have been correlated, and provide a rational approach to genetically directed DNA sequence analysis of genes 34--38 and their mutant variants that affect the assembly, structure and function of the tail fibers.

Plasmid ColIb (61.5 Mdal) was digested with restriction enzymes EcoRI and HindIII. The DNA digestion products were separated by electrophoresis on 1.2% agarose gels. There were identified 22 fragments of ColIb DNA generated by the endonuclease EcoRI and 21 fragments produced by HindIII. Molecular weights of the fragments were estimated. The total molecular weight of the fragments generated by EcoRI was 61.42 Mdal and for HindIII fragments 62.79 Mdal.

The transposon encoding resistance to mercuric ions, Tn501, is 5.2 (+/-0.1) x 10(6) daltons and is bounded by small inverted repeats. The restriction sites for the restriction endonucleases EcoRI, HindIII and SalGI have been mapped on the element.

The location of the rho gene and its position relative to the ilv genes of Escherichia coli K-12 was analyzed using genetic criteria, restriction enzyme cleavage, and maxicell analysis. Plasmids were constructed with deletions of the rho gene introduced in vitro, and lambda ilv-gal derivatives of lambda ilv-rho bacteriophage were isolated by recombination in vivo. A HindIII restriction fragment of 8 kilobases (kb) previously shown to contain at least part of the rho gene (Gray et al. 1981) was cloned into plasmid pMC81. This vector has transcription stop sites that present read-through expression of cloned genes from either direction, and cloning sites upstream of the lacZ gene coding for beta-galactosidase. The position of the rho gene and flanking sequences required for its expression were further localized to a region of approximately 2 kb by introducing deletions using restriction enzyme treatment of these plasmids. A promoter in the rho region was found to direct beta-galactosidase synthesis in these plasmid derivatives. Derivatives of lambda ilv-rho phage were isolated in vivo by pyrophosphate chelation selection for phage with reduced genome size. Restriction enzyme analysis of twelve of these derivatives revealed an unexpected bias towards phage recombinants as opposed to simple internal deletions.

Saccharomyces cerevisiae 2-mum DNA and some of its restriction fragments were integrated in vector pCR1 ,pBR313 or pBR322 and their expression in Escherichia coli P678-54 minicells was analyzed. 2mum DNA inserted at the EcoRI site of pCR1 or pBR313 and at the PstI site of pBR322 promoted the synthesis of polypeptides of 48,000, 37,000, 35,000 and 19,000 daltons. The DNA regions coding for these polypeptides were mapped on the 2-mum DNA molecule by insertion of single EcoRI or HindIII restriction fragments and comparison of the polypeptides produced. For the synthesis of the 37,000 dalton polypeptide, intact sites RIB and H3 were required. The disappearance of the 37,000 dalton polypeptide on interruption of one of these sites by insertion of the vector, was correlated with the appearance of a polypeptide of 22,000 or 23,500 daltons respectively. The DNA sequence coding for the 37,000 daltons polypeptide, therefore, has to be located in the S-loop region close to or overlapping with the site RIB and H3. Assuming that the 22,000 and the 23,500 dalton polypeptides are truncated forms of the 37,000 dalton polypeptide, the last polypeptide can be exactly mapped. The polypeptide of 48,000 daltons was mapped to that half of the L-loop segment containing the sites H1 and H2. If, however, HindIII fragment H1-H2 was expressed, the 48,000 dalton polypeptide was lost and concomitantly a 43,000 dalton polypeptide appeared. We assume that this polypeptide results from early termination of the polypeptide of 48,000 daltons. The 35,000 and 9,000 dalton polypeptides were mapped to the S-loop region. The integrated inverted repeat sequence of yeast 2-mum Dna did not induce any detectable insert-specific polypeptide synthesis.

A cleavage map of bacteriophage P1 DNA was established by reciprocal double digestion with various restriction endonucleases. The enzymes used and, in parenthesis, the number of their cleavage sites on the P1clts genome are: PstI (1), HindIII(3), BglII (11), BamHI (14) and EcoRI (26). The relative order of the PstI, HindIII and BglII sites, as well as the order of 13 out of the 14 BamHI sites and of 17 out of the 26 EcoRI sites was determined. The P1 genome was divided into 100 map units and the PstI site was arbitrarily chosen as reference point at map unit 20. DNA packaging into phage heads starts preferentially at map unit 92 and it proceeds towards higher map units. The two inverted repeat sequences of P1 DNA map about at units 30 and 34.

Three independent spontaneous mutations of prophage P1 affecting the ability of the phage to reproduce vegetatively are due to the insertion of a mobile genetic element, called IS 30. The same sequence is also carried in the R plasmid NR 1-Basel, but not in the parental plasmid NR 1. Southern hybridisation study indicates that the Escherichia coli K 12 chromosome carries several copies of IS 30 as a normal resident. IS 30 is 1.2 kb long and contains unique restriction cleavage sites for Bg/II, ClaI, HindIII, NciI and HincII, and it is cleaved twice by the enzymes HpaII and TaqI. The ends of IS 30 are formed by 26 bp long inverted repeats with 3 bases mismatched. Upon transposition IS 30 generates a duplication of only 2 bp of the target. The following observations suggest a pronounced specificity in target selection by IS 30. In transposition to the phage P 1 genome a single integration site was used three times independently, and in both orientations. A short region of sequence homology has been identified between the P 1 and NR 1-Basel insertion sites. IS 30 has mediated cointegration as well as deletion. The entire IS 30 sequences were duplicated in the cointegrates between a pBR 322 derivative containing IS 30 and the genome of phage P 1–15, and several loci on the P1–15 genome served as fusion sites, some of which were used more than once.

DNA preparations of the chloramphenicol resistance determining S. aureus plasmids pC194, pC223, and PUB112 can be fractionated by gel electrophoresis into various bands. Electronmicroscopic investigations of these various molecular species obtained with pC194 indicated that, depending on the preparations, 70 to 80% of the molecules were monomers, while the rest consisted of various classes of concatemeric and/or interlocked multimers. Measurements of the specific transforming activity of the various molecular classes indicated that the monomers had less than one thousandth the activity of the multimeric plasmic DNA. pC194 DNA of high specific transforming activity could also be obtained by ligation of HindIII generated monomers into concatemeric DNA.

The gltA gene from Escherichia coli, which encodes citrate synthase, has been located on a 3.24 Kb HindIII/EcoRl restriction fragment. This region contains one restriction site for BamHl and two for BglII. Defined restriction fragments from this region were cloned into suitably cleaved replicative form M13mp8 and M13mp9. The recombinants (M13gtlA1 leads to 10) were isolated as single stranded DNA and characterised on the basis of molecular weight and DNA sequence. The single stranded DNA was converted to the double stranded replicative form and used to transform E. coli strain JM103 from which bacteriophage were isolated. Infection of JM103 with different bacteriophage followed by measurement of expressed citrate synthase activity showed that the complete gltA gene must span the BamHl restriction site, that the control region was on the 5'-terminal side of this restriction site and that the coding region for citrate synthase protein commenced on the 3'-terminal side. Analysis of the DNA sequence of this region allowed us to confirm this model, to identify the start sequence for translation of the structural gene and a number of sequences controlling the initiation of transcription. Of special interest is the fact that there must be an extensive leader sequence (305 nucleotides) separating the predicted sites for initiation of transcription and translation.

Comparative restriction cleavage analysis of the genomes of bacteriophage P7, of several recombinant phages between P7 and P1, and of bacteriophage P1 allowed to draw PstI, BglII, BamHI and HindIII cleavage maps of all genomes studied. The data obtained complement Yun and Vapnek's (1977) conclusions with regard to areas of major nonhomology based on electron microscopical heteroduplex analysis and they identify several additional minor differences between P1 and P7. The use of hybrid phage strains allowed to locate the genes for particular functions on the physical genome map.

The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of beta-lactamase production. By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that approximately 1.9 X 10(6) daltons of the 6.0 X 10(6) dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.

Two structural variants of the rat alpha-fetoprotein (AFP) gene have been detected in different inbred strains of rats by EcoRI or HindIII restriction enzyme cleavage of cellular DNA, agarose gel electrophoresis and Southern blot hybridization using 32P-labeled cloned rat AFP cDNA probes. The type I AFP gene variant is characteristic of the Sprague-Dawley strain, and type II is found in Buffalo rats. These variants appear to represent two different allelic forms of the rat AFP gene since they are inherited in a normal Mendelian fashion when Sprague-Dawley and Buffalo rats are crossed. The mapping results suggest that the two allelic variants differ from each other by multiple cleavage site variations (base pair substitutions) and by an insertion or deletion of DNA sequences. An extensive sequence variation appears to exist between the two forms of the rat AFP gene; we have estimated that as much as 2.7% of the nucleotides in this region vary between the two alleles.

A map of cleavage sites for restriction endonucleases EcoR1, BamHI, HindIII, and SalI on Tn2603, a transposon encoding resistance to ampicillin, streptomycin, sulfonamide, and mercury, was constructed by an analysis of restriction cleavage patterns of plasmid pMK1.:: Tn2603 and its deletion derivative. By cloning the fragments generated from pMK1::Tn2603 with these restriction endonucleases to a pACYC184 plasmid vehicle, the regions necessary for expression of resistance were located on the restriction cleavage map of Tn2603. Ampicillin, streptomycin, and sulfonamide-resistance genes were mapped in a cluster on the region between the center and the right and the mercury-resistance gene was located to the left of the map. The final functional map of Tn2603 was compared with those of Tn4 and Tn21 and the evolutional relationships between them were discussed.

Replicating DNA molecules of the mini R6-5 plasmid, pKTO71, were purified by equilibrium centrifugation in two successive ethidium bromide-caesium chloride gradients, converted to linear forms by cleavage with either HindIII or BglII restriction endonuclease, and examined in the electron microscope. Determination of the replication fork positions in 65 replicating molecules demonstrated that replication is initiated at a unique location on the plasmid and that it proceeds uni-directionally from this site. The direction of replication is such that the origin-proximal BglII cleavage site is replicated late or, in the case of the parent R6-5 plasmid, is such that the R-determinant region of the molecule is replicated early. The origin of replication, located by these experiments at R6-5 coordinate 98.6 kb, is clearly distinct from that of the R6-5 incompatibility determinant which has been shown to be located on an adjacent PstI-generated DNA fragment whose termini have R6-5 coordinates 96.8 and 97.9 kb. This result indicates that the incompatibility function is not an origin DNA sequence.

When E. coli cells carrying the plasmid pLG13 (coding for the newly discovered type II restriction endonuclease EcoRV) are infected with phage T3 or T7, only T7 is able to replicate normally. T3 wild-type as well as its ocr- mutants are subject to DNA restriction in vivo and in vitro. The EcoRV enzyme cuts T3 DNA at 5 sites. T7 and its ocr- mutants have no EcoRV sites in their DNA. In contrast to the anti-restriction activity of the T3 and T7 ocr+ gene function against type I and III restriction enzymes, the ocr+ protein is unable to inactivate the type II restriction endonuclease EcoRV.

The cleavage map of the plasmid RK2 was determined for the five restriction endonucleases EcoRI, HindIII, Bam H-I, Sal I and Hpa I. DNA has been inserted into several of these sites and cloned in Escherichia coli. Efforts to obtain derivatives of RK2 reduced in size by restriction endonuclease digestion of the plasmid were not successful and indicated that genes required for the maintenance of this plasmid in E. coli are not tightly clustered. An RK2 derivative possessing an internal molecular rearrangement was obtained by transformation with restriction endonuclease digests of the plasmid.

The glnA, ntrB and ntrC genes of Klebsiella pneumoniae have been cloned, on a 12 kb HindIII fragment, into the plasmid pACYC184. In a coupled in vitro transcription/translation system the resultant plasmid, pGE100, directed synthesis of five polypeptides (molecular weights 73, 53, 51, 39, 36 kd) from the cloned fragment. A number of plasmids were derived from pGE100 and studied by complementation analysis and in vitro transcription/translation in order to locate particular genes and identify their products. On the basis of the results presented here, together with previous genetic and physical characterisation of the glnA gene and its product in other enteric bacteria, we propose that the 53 kd polypeptide is the glnA gene product (glutamine synthetase monomer). Two polypeptides (36 kd and 51 kd) were synthesised from a 3 kb region previously defined as glnR. In E. coli and S. typhimurium this region comprises two genes ntrB and ntrC with products of 36 kd and 54 kd respectively. This analogy supports the idea that the 36 kd and 51 kd polypeptides are the products of the K. pneumoniae ntrB and ntrC genes respectively. Comparison of these assignments with the physical map of the region indicates a gene order glnA, ntrB, ntrC. Assessment of the Nif phenotype of a glnA-ntrC deletion strain carrying various clones from pGE100 demonstrated that glnA is not required for expression of the nif regulon and that of the three genes cloned, ntrC alone is sufficient for nif expression.

The Ti plasmid DNA maintained in octopine-type crown gall tumor lines is variable, but always includes at least part of the Ti plasmid that maps over the region of Hind III fragment 1 of pTi-B6-806. The right-hand boundary of transferred DNA (T-DNA) varies considerably among the three independent tumor lines examined; the left boundary was not located definitively. The T-DNA of two sibling clones of the same tumor line, E1 and E9, appears identical. The copy number of T-DNA in E9 tumor DNA appears higher for the right end (about 30 copies) than for the left end (approximately 1 copy).