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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.

An iodometric assay for β-lactamase has been employed for identifying colonies of Escherichia coli transformed to tetracycline resistance (Tcr) by pBR322 carrying inserts at the PstI site. This assay is based upon the ability of β-lactamase produced by ampicillin-resistant (Apr) cells to convert penicillin to penicilloic acid which in turn binds iodine. Growth and selection of E. coli transformed to AprTcr or ApsTr are obtained on Luria agar plates containing soluble starch and tetracycline. When indicator solution containing penicillin and iodine is added to the colonized plates, β-lactamase-producing (Apr) colonies rapidly clear the overlying indicator solution whereas non-β-lactamase-producing (Aps) colonies exhibit no clearing effect. This reaction persists and substantial numbers of viable cells remain well beyond the end of the 15-min observation period. In post-test assessment of phenotype, all nonclearing colonies exhibited the ApsTcr phenotype while those that cleared the indicating solution exhibited the AprTcr phenotype. Application of this assay to an actual transformation experiment permitted rapid and unambiguous identification of the ApsTcr phenotype.

Narcolepsy is a sleep disorder completely associated with HLA-DR2,Dw2. We demonstrated the 100% presence of three DQ beta fragments (EcoRI 2.4-kb, BamHI 2.9-kb, and PstI 12-kb) in narcoleptic patients that were detected in healthy DR2 controls only at decreased frequencies. In this paper, we have cloned the DQ beta gene from three Japanese narcoleptic patients and sequenced their beta 1 domain in order to study the sequence polymorphisms that might exist in the DQ beta genes of patients, but no difference in sequence could be found between narcoleptic and healthy individuals, suggesting that narcolepsy is not due to mutation in the DQ beta gene. In this context, a possible role of the HLA class II antigens in narcolepsy is discussed.

The sequence of part of the larger unique region of the yeast 2 micron plasmid cloned in pMB9 has been determined. The sequence extends from the single EcoRI site in this region to the AvaI site and includes the single PstI site and HpaI site. A notable feature of this sequence is the presence of tandem repeats of 124 residues beginning at the HpaI site and extending beyond the AvaI site. The sequence was determined independently by both the Maxam-Gilbert procedure applied to isolated restriction fragments, and by the chain-termination procedure applied to restriction fragments cloned in the single-stranded phage M13mp2 and purified by plaque selection.

Summary Horse DNA samples digested with PstI and probed with the rabbit β1 globin gene show three phenotypes determined by one fragment of variable length (about 5·1 or 3·3 kb). Family data demonstrate that these fragments segregate as Mendelian alleles. The frequencies of the two alleles are 0·66 for the 3·3-kb fragment and 0·34 for the 5·1-kb one. Another polymorphism has been detected with BamHI. Again three phenotypes determined by two alleles (fragments of 7·5 and 3·8 kb) have been observed. Allelic frequencies of the 7·5- and 3·8-kb fragments are 0·24 and 0·76 respectively. The two polymorphic sites are non-randomly associated.

Polysomal poly(A)RNA was transcribed by avian myeloblastosis virus (AMV) reverse transcriptase and double-stranded complementary DNA (cDNA) was then synthesized and cloned in Escherichia coli C600 by (dG)-(dC) tailing in the PstI site of pBR322. A bank of mRNA sequences from maturating soybean seeds (average weight 200 mg) was obtained. (32)P-cDNA based on mRNA from various organs of the plant (leaves, axes, seeds) was synthesized. The clones were hybridized with different kinds of (32)P-cDNA on nitrocellulose filters. Approximately 13% of the clones were specific for the maturating stage, and more than a half the clones gave a positive result with all kinds of cDNA.

A cosmid library of copper-resistant (Cur) Pseudomonas syringae pv. tomato PT23 plasmid DNA was constructed and mobilized into the copper-sensitive recipient P. syringae pv. syringae PS61. One resultant cosmid clone, pCOP1 (46 kilobases), conferred copper resistance. The PT23 Cur gene(s) was located on pCOP1 by subcloning PstI restriction endonuclease fragments of pCOP1 in the broad-host-range vector pRK404. A subclone containing a 4.4-kilobase PstI fragment conferred Cur on PS61. The Cur gene(s) was further located by insertional inactivation with Tn5. A subcloned fragment internal to the Cur determinant on pCOP2 was probed to plasmid and chromosomal DNA of four copper-resistant and three copper-sensitive strains of P. syringae pv. tomato. The probe hybridized to plasmids in resistant strains, but showed no detectable homology to copper-sensitive strains.

Cleavage of pBR322 DNA I by the restriction endonuclease HinfI is preferentially inhibited at specific HinfI cleavage sites. These sites in pBR322 DNA I have been identified and ordered with respect to the frequency with which they are cleaved. The HinfI site most resistant to cleavage in pBR322 DNA I is unique in that runs of G-C base pairs are immediately adjacent on both sites. Two differently permuted linear (DNA III) species were produced by cleavage with two different restriction endonucleases, PstI and AvaI. Only one of these linear molecules, that produced by PstI, exhibits the same preferential cleavage pattern as DNA I. The second linear species, that arising from AvaI digestion, shows pronounced relative inhibition of cleavage at the HinfI sites nearest the ends of the molecule (100 to 120 base pairs away, respectively). This result suggest that proximity to the termini of a linear DNA molecule might also influence preferential cleavage. The possibility of formation of stem-loop structures does not appear to influence preferential cleavage by HinfI.

Bovine pancreas contains two polypeptide trypsin inhibitors which are not homologous and differ in their inhibitory activity towards chymotrypsin, kallikrein, elastase and other serine proteinases [1]. The Kunitz inhibitor [2, 3] and the Kazal inhibitor [4, 5] are present in approximately equimolar concentrations in bovine pancreatic tissue [6], yet only the Kazal inhibitor is detectable in the pancreatic juice [6, 7]. The Kazal inhibitor has been named the pancreatic secretory trypsin inhibitor, PSTI [8] because its concentration in the pancreatic juice parallels that of the exocrine secretory proteins [7, 9, 10]. The Kunitz inhibitor is considered the “intracellular” inhibitor [7]. However, no direct information is available concerning the intracellular localization of these inhibitors in the pancreas. A sensitive and specific analytical method for the measurement of Kazal and Kunitz inhibitors at the picogram-nanogram level is required for intracellular distribution studies. The preparation of trace labelled 131I-Kunitz inhibitor by the chloramine T method and its use in a radioimmunoassay have been described by Arndts et al. [11]. In this communication we report the preparation of 125I derivatives of Kazal and Kunitz inhibitors by the lactoperoxidase method and present a radioimmunoassay for each inhibitor.

Pancreatic secretory trypsin inhibitor (PSTI) has been thought to be only a secretory trypsin inhibitor of human pancreas, but the serum content of immunoreactive PSTI is elevated without pancreatic disease. Using the peroxidase-antiperoxidase method, immunoreactive cells for PSTI were found in human pancreas, stomach, duodenum, appendix, colon and urinary tract of both fetus and adult, adult gall bladder, and fetal lung. PSTI-immunoreactive cells were identified in fetal pancreas at the tenth gestational week, and in extrapancreatic tissues at the sixteenth (gastrointestinal and urinary tract) and twentieth weeks (lung). PSTI-immunoreactive cells of fetal lung were present in neuroepithelial bodies. Strongly positive cells in fetal duodenum were argyrophilic and resembled endocrine cells. Immunohistochemical study was also performed on tissues associated with inflammatory diseases of gastrointestinal tract. The distribution pattern of immunoreactive cells in the stomach varied in accordance with chronic gastritis. Immunoreactive cells were also found in endocrine micro-nests and in a carcinoid tumor associated with fundic gastritis. These results suggest that PSTI may play some physiological role other than secretory trypsin inhibition of the pancreas.

The mutagenic and carcinogenic substance benzo[a]pyrene reacts with DNA following activation to its corresponding 7,8-diol 9,10-epoxide (BPDE), and the major DNA adduct (BP-N2-Gua) is formed when the C(10)-position of BPDE reacts with the N2-position of guanine. It is unknown if this adduct is a premutagenic lesion in vivo. Herein, the construction and characterization of an M13mp19-based, E. coli vector that contains BP-N2-Gua located in the unique PstI restriction endonuclease recognition site at nucleotide position 6249 in the (-)-strand is described (designated, BP-N2-Gua-M13mp19). First, the oligonucleotide 5'-TGCA-3' was reacted with BPDE and a product (5'-T(BP-N2)GCA-3') was isolated by HPLC that, when enzymatically digested to deoxynucleosides, yielded an adduct that comigrated on HPLC with an authentic BP-N2-Gua deoxynucleoside standard. Second, the 5'-hydroxyl group of 5'-T-(BP-N2)GCA-3' was phosphorylated with ATP and T4 polynucleotide kinase, and the product (5'-pT(BP-N2)GCA-3') was purified by HPLC. This product is stable when heated at 80 degrees C at both neutral and alkaline pH. Third, M13mp19 was manipulated such that the sequence 5'-pTGCA-3' was selectively removed from the (-)-strand in its unique PstI recognition site, and 5'-pT(BP-N2)GCA-3' was ligated into this gap with T4 DNA ligase and ATP. The product of this reaction (BP-N2-Gua-M13mp19) was shown to be insensitive to cleavage by PstI, which suggests that a modification is located in the PstI recognition site. The most likely modification is the adduct BP-N2-Gua.

The capsule plasmid pTE702 of Bacillus anthracis has been physically mapped with the restriction endonucleases HindIII, PstI, BamHI, SalI, and XhoI. A HindIII fragment map of pTE702 (96.5 kb) was obtained by analysis of the recombinant plasmids and cosmids containing overlapping fragments partially digested with HindIII. The physical map for PstI, BamHI, SalI, and XhoI was obtained by double digestion mapping of these sites in relation to the HindIII sites. The replication region of pTE702 was determined by in vitro genetic replicon labeling in B. subtilis.

A cDNA clone for mouse pro alpha 1(I) collagen has been isolated and sequenced. A 1.8-kb PstI fragment spans nucleotides -1991 to -159 of the alpha-domain of mouse pro alpha 1(I) collagen.

Unintegrated linear and closed circular DNAs of B- and N-tropic endogenous BALB/c murine leukemia virus (MuLV) were extracted from newly infected mouse cells and cleaved with EcoRI, XhoI, PvuI, HindIII, SalI, XbaI, KpnI, SmaI, and PstI restriction endonucleases. The DNA fragments were separated by electrophoresis and analyzed by the Southern blot hybridization procedure. EcoRI did not cleave the two genomes. A physical map of 15 cleavage sites on B- and N-tropic genomes was constructed with the other restriction endonucleases. Identical cleavage sites of B- and N-tropic MuLV DNAs were found with all these enzymes. However, the N-tropic linear genome was found to lack about 75 base pairs at each end of the molecule. PstI, KpnI, and SmaI recognize a cleavage site at both ends of the linear molecules. And sequences derived from the 5' end of the RNA genome were found in the third left end of the linear DNA and at its extreme right-end terminus, suggesting the presence of redundant sequences. Two species of closed circular viral DNA were observed. The larger species has the same size as the linear molecule and appears to be a circularized form of linear DNA. The smaller species contains sequences common to both the linear and the larger circular viral DNA but seems to be deleted from sequences present at either one or both ends of the linear DNA. Therefore, the general structure of the linear and circular DNA species of these B- and N-tropic endogenous BALB/c MuLV appears analogous to the structure found with other retroviruses.

The temperature dependence of the T4 DNA ligase-catalyzed joining of plasmid DNA linearized by the action of HaeIII, EcoRI and PstI restriction endonucleases has been investigated by electron microscopy analysis. The extent of joining is maximal at 4 degrees and decreases with increasing temperatures following sigmoid-like curves. The temperature at which 50% of the maximal reaction is still observable increases going from DNA termini without single-stranded overlaps (produced by HaeIII) to termini with four nucleotides overlap, composed only by two A and two T (produced by EcoRI) to termini with four nucleotide overlap, composed by A, T, G and C (produced by PstI).

Polymorphism in restriction site cleavage (PstI, SphI, PvuII, HindIII) has been noticed in several occasions in the telomeric sequences harbouring trypanosome variant-specific antigen genes (1, 2, 3). This polymorphism has been further investigated and seems best interpreted as due to partial DNA modification in GC dinucleotides. The actively transcribed telomeric genes do not exhibit such a polymorphism; furthermore, in at least three independent cases, gene inactivation is linked to the appearance of polymorphism. It could thus be hypothesized that DNA modification prevents antigen gene transcription, or vice-versa. We report however that at least some telomeric antigen-specific sequences of the procyclic trypanosomes (in vitro culture form) are not polymorphic, although they do not synthesize any variant-specific antigen mRNA. There is thus no absolute relationship between the absence of polymorphism and antigen gene transcription.

Two types of cDNA, which code for the two types of rat pancreatic secretory trypsin inhibitors (PSTIs), were cloned and sequenced. Both predicted amino acid sequences consisting of 79 amino acids, with the secretion signal peptide consisting of 18 and 23 amino acids for PSTI-I and PSTI-II, respectively. The nucleotide sequences were 91% homologous between the two cDNAs, but 68% and 65% homologous, respectively, when compared with human PSTI cDNA. Northern blot analyses showed that PSTI-I is expressed in the pancreas, whereas PSTI-II is expressed in the pancreas and the liver using the same promotor. Southern blot analyses suggested that both PSTI-I and PSTI-II genes are single copy genes per haploid genome. Duplication of rat PSTI gene seems to have occurred recently, after the divergene of humans and rats.

Summary. A cDNA library for porcine skeletal muscle was established in the vector pBR322. The library was screened with an oligonucleotide probe coding for a hexapeptide from glucosephosphate isomerase (Gpi). A positive clone with an insert of about 450 bp and restriction sites for PstI, BamHI and PvuII was isolated. A 362-bp PstI fragment was sequenced and shown to contain the codons for the hexapeptide as well as the remaining 29 amino acids of this Gpi peptide. The PstI fragment was used to probe pig genomic DNA. The restriction enzymes PvuII and SacI detected a set of polymorphisms with five bands, behaving as a set of insertion/deletion polymorphisms.

The cleavage sites of the restriction nucleases HpaI, HpaII, HindII, HindIII, and PstI have been mapped on the DNA of the bacteriophage PM2. This map has been used for the localization of two strong binding sites of Escherichia coli RNA polymerase on PM2 DNA.

In order to facilitate the production of the B subunit of the heat- labile enterotoxin of E. coli (LT-B) for use as an immunologic carrier, we utilized recombinant DNA technology to construct a plasmid containing the genes for production of the B subunit only from a human isolate of enterotoxigenic E. coli. the 0.8kb gene fragment encoding synthesis of LT-B was cloned into plasmid pBR322 following sequential digestion of the enterotoxin plasmid with restriction endonucleases PstI and HindIII. Cloned B subunit was isolated from cell lysates in its oligomeric form, was structurally identical to native B subunit when examined by SDS-PAGE, immunologically identical in ELISA, and contained no demonstrable A subunit in either assay. Depsite deletion of the A subunit, cloned LT-B did induce morphologic alterations in cultured mouse Y1 adrenal cells at high concentrations, an indication of residual toxicity. The 0. 8kb gene fragment was recloned into the plasmid vector pUC8 and this resulted in a construct which coded for synthesis of LT-B free of biologic activity but still capable of binding to the epithelial cell surface. Peptide fragmentation resulted in loss of binding activity, suggesting a conformational binding determinant.

A decanucleotide dC-C-C-T-C-G-A-G-G-G was synthesized by phosphotriester block condensation. Three protected blocks, dCCCTp, dCGAp and dGGG were prepared using N-acyl, 5'-O-monomethoxytrityldeoxynucleosides as the starting materials. The protected dGGG which served as the 3'-terminal block was synthesized by condensation of 3'-O-benzoyl-N-isobutyryldeoxyguanosine (5'-hydroxy component) with 5'-O-monomethoxytrityl-N-isobutyryldeoxyguanosine 3'-O-p-chlorophenyl phosphate (3'-O-phosphodie ster component) using mesitylenesulfonyl triazolide as the condensing reagent followed by removal of the 5'-monomethoxytrityl group for repeated condensation. The other two blocks were prepared by using the 3'-O-p-chlorophenyl phosphoranilidate of N, 5'-protected deoxynucleosides as the 3'-end unit (5'-hydroxy component). The phosphoranilidate of the fully protected trimers was removed by treatment with isoamyl nitrite for the condensation with the 5'-hydroxyl group of the growing chain. Fully protected nucleotides were isolated by chromatography on silica gel and the deblocked product was purified by ionexchange chromatography on DEAE-cellulose. The decanucleotide was characterized by mobility shift analysis and complete enzymatic digestions after labelling the 5'-end with [γ-32P] ATP using polynucleotide kinase.

Xanthomonascampestris pv. glycines causes bacterial pustule disease on susceptible soybean cultivars. This disease is characterized by host cell hypertrophy leading to pustule formation at the site of infection. Sixteen virulent and four avirulent isolates were examined for the presence of plasmids by detergent lysis and electrophoresis. All virulent strains harbor between one and three plasmids. All avirulent strains are plasmid free. Ten of the virulent strains contain an approximately 18 megadalton (mdal) plasmid. The other six virulent strains have a plasmid between 11 and 29 mdal in size. Restriction endonuclease digestion with BamHI and PstI indicates identity or near identity for all these plasmids. Two virulent strains harbor an additional 26 mdal plasmid. One of these also contains a 110 mdal plasmid. Nine virulent strains have in common an approximately 1 mdal plasmid which is, in all but one case, refractory to BamHI and PstI digestion. None of the strains are resistant to any of the 15 antibiotics and antibacterials examined. The function of these plasmids in X. campestris pv. glycines is not known. The presence of the 18 mdal plasmid or its derivatives in all virulent strains, and its absence from all avirulent strains tested to date, suggests a role in pathogenicity.

14 conjugative plasmids originated from different bacterial hosts and geographical sources were identified as members of the incompatibility group IncX with R6K as the reference plasmid. In spite of their close genetic relatedness (transfer and incompatibility properties), their molecular sizes ranged between 39 and 77 kb and DNA fingerprinting with endonucleases such as EcoRI, EcoRV, BamHI, Bg/I, Bg/II, PstI and Sa/I revealed a great variety of fragment patterns.

Publisher Summary This chapter focuses on preparing recombinants, DNA fragments can be joined to plasmid or virus vectors in a number of ways. Vectors linearized with restriction enzymes can be annealed to DNA fragments terminating in homologous sequences. Alternatively, vectors and DNA fragments can be terminated in the complementary oligodeoxynucleotides oligo(dA) and oligo(dT) or oligo(dG) and oligo(dC). With the use of the plasmid pBR322 linearized with the restriction enzyme PstI, the addition of oligo(dG) restores the PstI site. This conveniently permits the excision of the inserted DNA sequence from the recombinants. The PstI site is within the β-lactamase gene, so that these pBR322 recombinants with inserts are penicillinsensitive, but retain their tetracycline resistance. The transformation efficiency of χ1776 by intact plasmid pBR322 by the above procedure yields approximately 1 to 3×10 6 transformants per microgram of pBR322 DNA.

A total of 74 strains out of 33 strains of adenovirus type 19 (Ad19) plus 103 strains of type 37 (Ad37) isolated from patients with conjunctivitis at two ophthalmology clinics in Hiroshima City during the period March 1983 to December 1986 were analyzed by eight DNA restriction endonucleases in comparison with their prototype strains. All 27 Ad19 isolates examined displayed identical DNA cleavage patterns with all enzymes used (HindIII, KpnI, PstI, XhoI, BamHI, SacI, EcoRI, and SmaI), but their cleavage patterns were different from those of the prototype except with HindIII. The genome type of these isolates was tentatively named Ad19a. Forty-seven Ad37 isolates examined were divided into three genome types. They were tentatively named Ad37p, Ad37a, and Ad37b: 16 isolates (Ad37p) displayed DNA cleavage patterns identical with those of the prototype with all eight enzymes described above. Thirty isolates (Ad37a) showed the same patterns as the prototype except with EcoRI. One isolate (Ad37b) showed the same patterns as the prototype except with SmaI. The most frequently isolated genome type during the period studied was Ad37a, but the change of the predominant genome type in yearly incidences was observed.

The interaction of AOT reverse micelles with Met-enkephalin, the pancreatic secretory trypsin inhibitor (PSTI) and the epidermal growth factor (EGF) is examined by NMR methods and the three systems are compared. While Met-enkephalin adopts a folded conformation, PSTI appers to become highly flexible, suggestive of a non-specific interaction with the micelles. On the other hand, the EGF spectrum shows that, although the main globular features of the protein are retained in the presence of AOT, the C-terminal fragment has to rearrange its conformation when put in contact with the micelle wall.

Pancreatic secretory trypsin inhibitor (PSTI) was purified from canine pancreatic juice by HPLC. Canine PSTI inhibited bovine trypsin activity stoichiometrically and strongly with a dissociation constant of below 10 −9 M. The amino acid sequence of canine PSTI was determined by conventional methods. It had one more amino acid residue at the amino-terminus than other mammalian PSTIs, i.e. human, porcine, bovine and ovine.

The effect of temperature and pH on the association equilibrium constant (Ka) for the binding of the bovine pancreatic secretory trypsin inhibitor (bovine PSTI, type I; Kazal inhibitor) to bovine β-trypsin, bovine α-chymotrypsin and bovine trypsinogen has been investigated. The results suggest that serine (pro)enzyme inhibitor interaction involves both rigorous spatial configuration and molecular flexibility.

1. We have determined the frequency of DNA polymorphisms of the human apolipoprotein AI-CIII-AIV gene cluster, detected with XmnI, PstI, and PvuII, in a group of patients with peripheral arterial disease. 2. Of the patients, 81 had no evidence of disease in the coronary and carotid arteries, 73 had coronary artery disease but no evidence of carotid artery disease, 25 patients had carotid artery disease but no evidence of coronary artery disease, and 38 had both coronary and carotid artery disease. 3. Levels of triacylglycerol, cholesterol, apolipoprotein B and apolipoprotein AI were not significantly different between the four patient groups. 4. The frequencies of the alleles for the apolipoprotein AI-CIII-AIV polymorphisms, detected with XmnI, PstI and PvuII, did not differ significantly in the patient groups when compared with a sample of clinically well normolipidaemic individuals also from a London population. 5. All five patients with the XmnI genotype we designate X2X2 had high levels of cholesterol, apolipoprotein B and apolipoprotein AI. 6. Patients with the rare VB2 allele of the apolipoprotein CIII-AIV restriction fragment length polymorphism had lower levels of cholesterol, acylglycerol and significantly lower levels of serum apolipoprotein. 7. Our observations suggest that variation in the apolipoprotein AI-CIII-AIV gene cluster may not be contributing significantly to the development of peripheral arterial disease, but variation associated with some of the restriction fragment length polymorphisms may be involved in determining levels of cholesterol- and apolipoprotein-B-containing lipoproteins.

Cloned cDNAs for rat liver serine:pyruvate aminotransferase were obtained by screening of a cDNA expression bank of rat liver with an antibody against the enzyme. Nineteen clones were isolated from 33000 transformants and most of them had common fragments of cDNA on analysis by digestion with some restriction enzymes. These clones were identified as those containing cDNA for serine:pyruvate aminotransferase by the following criteria. (a) At the nucleic acid level, a 500-base-pair fragment of cDNA prepared by digestion of cDNAs with EcoRI and PstI hybridized with the mRNA coding for serine:pyruvate aminotransferase as judged by hybrid-selected and hybrid-arrested translations. (b) Specific proteins were detected in nine bacterial clones, a 40-kDa protein in one clone and a 39-kDa protein in eight clones. Among them only the 40-kDa protein was found to be solubilized from the cell by sonication, and this protein was immunoprecipitated with an antibody against serine:pyruvate aminotransferase of rat liver. (c) High activity of serine:pyruvate aminotransferase was expressed both in whole cell suspension and sonicated extract prepared from the transformant producing the 40-kDa protein, and 99% of the activity was immunoreactive with the antibody. Two types of mRNA for serine:pyruvate aminotransferase were detected on the RNA blot analysis by using cloned cDNA fragment as a probe. The larger mRNA (approximately 1600 nucleotides) was glucagon-inducible while the smaller one (approximately 1500 nucleotides) was not affected by the hormone.

Using the restriction endonucleases Xho I, Sac I and Pst I, physical maps of Spirodela oligorrhiza (duck weed) chloroplast DNA [with a relatively large m. wt. of 120 Md; see van Ee et al. (1980)] were constructed. The overall structural organization of S. oligorrhiza chloroplast DNA appears to be rather similar to that of chloroplast DNAs of other higher plants. It is composed of two invertedly repeated DNA segments of 17 Md, separated by a small single-copy region of 19 Md and a large single-copy region of 69 Md. Apparently the extra DNA (as compared with chloroplast DNAs of other higher plants) is present within the large single-copy region.

This paper is a survey of our recent work concerning the dynamics of the association of trypsin and chymotrypsin with the pancreatic trypsin inhibitors, that is, the Kunitz inhibitor (PTI)1 and the Kazal (secretory) inhibitor (PSTI).

A restriction endonuclease cleavage map is presented for mouse mitochondrial DNA. This map was constructed by electron microscopic measurements on partial digests containing fixed D-loops, and by electrophoretic analysis of partial and complete single enzyme digests, and of double digests. No map differences were detected between mitochondrial DNA from cultured LA9 cells and an inbred mouse line for the six endonucleases used. Three cleavage sites recognized by HpaI, five sites recognized by HincII, two sites recognized PstI and four sites recognized by BamI were located with respect to the origin of replication and the EcoRI and HinIII sites previously determined by others. No cleavages were produced by KpnI or SalI. The migration of linear DNA with a molecular weight greater than 1 X 10(6) was not a linear function of log molecular weight in 1% agarose gels run at 6.6 volts/cm.

Ten restriction fragment length polymorphisms of the LDL receptor gene were used for haplotype analysis in 12 unrelated patients with homozygous familial hypercholesterolemia. These patients were drawn from the Black, Coloured, and White population groups and collectively represent 24 mutant alleles underlying the FH phenotype. Five distinct haplotypes were detected. Hybridization analysis using DNA codigested with EcoRI and PstI revealed that haplotype IV was associated with two distinct mutations. When coupled to the recent demonstration by other workers of two receptor defects in South African Afrikaners homozygous for FH and haplotype I, these data are suggestive of at least seven distinct LDL receptor mutations in the FH patients examined and thus in the general South African population.

Fragments were isolated from subclones containing the insulin receptor cDNA described. Probe 1 as obtained from an SP64 subclone containing the 1011bp EcoRI fragment from the 5{prime} region of the U11rich cDNA. Probe 1 was a 677bp XhoI/EcoRI fragment from the {alpha} subunit region of the IR cDNA corresponding to nucleotides 334 to 1011, the putative ligand binding domain. Probe 2 was obtained from an SP64 subclone containing the 4190bp EcoRI fragment from the 3{prime} end of the U11rich cDNA. Probe 2 was a 1599 bp PstI fragment from the {beta} subunit region of the insulin receptor cDNA corresponding to nucleotides 2746 to 4345, encoding the tyrosine kinase domain. Segregation in at least one family was observed for the PstI, KpnI, and RsaI ({beta}) polymorphisms.

Complexes of intact nuclear DNA with proteins undisssociable by 2.0 M NaCl and nonionic detergents were analyzed by agarose gel electrophoresis following physical or enzymatic fragmentation. Sulfhydryl molecules converted these DNAs (but not the bacteriophage λ DNA) into smaller-Mr forms. Following limited restriction endonulease digestion of complexes with PstI most of the nuclear DNA formed a high-molecular-mass band in the 60–110 kbp range. These 60–110 kbp fragments, releasable from the rest of nuclei by sulfhydryl molecules, have similar sizes to nuclear DNA loops detected by other techniques and may derive from supranucleosomal organizational units in the chromatin complex.

Using alternating poly(dG-dC) · poly(dG-dC) and electron microscopy (EM), a method has been developed for detecting regions of Z conformation in DNA preparations. The procedure was developed with poly(dG-dC) · poly(dG-dC) which had been converted to the Z conformation with MnCl2 and mild heat treatment. Conditions were found for reaction of this DNA with polyclonal anti-Z antibodies from rabbit, and further reaction of this mixture with gold-labelled anti-rabbit antibodies from mouse. Spreading of these samples onto air-water interfaces and examination by EM revealed gold particles aligned along strands of poly(dG-dC) · poly(dG-dC). The method was refined and simplified using monoclonal antibodies and tested with the 2.2-kb plasmid, pDHg16, carrying a single tract of alternating d(G-C)23. Treatment with MnCl2 and mild heat was not necessary, as the superhelicity of this molecule ensured that the d(G-C) tract was in the Z conformation. Conditions were found for successful conjugation of mouse monoclonal anti-Z antibodies with colloidal gold (G10), 10.7-nm average diameter. The conjugate was then reacted with superhelical pDHg16, stabilized in polyethylene glycol and cross-linked with glutaraldehyde. Examination by EM showed gold particles at one site on the negatively superhelical circular DNA molecule. When these molecules were linearized with PstI, gold particles were found to occur at an average position 35% ± 3% from one end. This location agrees well with the known position of the center of the alternating d(G-C) tract with respect to the PstI restriction site (36.8%).

All but one of the PstI restriction fragments from mung bean, pea, and spinach chloroplast DNAs have been stably cloned into pBR322. Large fragments (15–54 kb) were cloned at low efficiencies which decreased with increasing fragment length. However, plasmids containing fragments above 25–30 kb were too unstable to be useful. In particular, pBR322 derivatives containing the largest mung bean and spinach fragments (34 kb and 54 kb, respectively) are extremely unstable and rapidly delete parts of the plasmid sequence. The SalI fragments of mung bean chloroplast DNA which cover the 34-kb PstI fragment have been cloned into pBR322. Similarly, the XhoI fragments of spinach chloroplast DNA which cover all but 0.9 kb of the 54-kb PstI fragment have been cloned into pACYC177. After a search of several thousand recombinants we were unable to recover a clone containing a 12.2-kb pea chloroplast PstI fragment and suggest that some property of its sequence may be inimical to the cloning process. The identity of the cloned fragments to native chloroplast DNA restriction fragments is demonstrated by restriction analysis and by the ability to construct detailed restriction maps of the mung bean and pea chloroplast genomes.

Restriction endonuclease cleavage maps for BamHI, EcoRI, HindIII, PstI, and SalI have been determined for the Escherichia coli bacteriophage BF23 DNA.

A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 has been constructed. In one of these vectors a kanamycin-resistance marker has been inserted into the center of the symmetrical MCS7 to yield a restriction-site-mobilizing element (RSM). The drug-resistance marker can be cleaved out of this vector with any of the restriction enzymes that recognize a site of the flanking sequences of the RSM to generate an RSM with either various sticky ends or blunt ends. These fragments can be used for insertion mutagenesis of any target molecule with compatible restriction sites. Insertion mutants are selected by their resistance to kanamycin. When the drug-resistance marker is removed with PstI, a small in-frame insertion can be generated. In addition, two new MCSs having single restriction sites have been formed by altering the symmetrical structure of MCS7. The resulting plasmids pUC8 and pUC9 allow one to clone doubly digested restriction fragments separately with both orientations in respect to the lac promoter. The terminal sequences of any DNA cloned in these plasmids can be characterized using the universal M13 primers.

Three classes of phosphotransferase system mutants in Salmonella typhimurium were selected through their resistance to 3-deoxy-3-fluoro-D-glucose (DFG). Strains with mutations in the ptsH (HPr) and/or pts I (enzyme I) genes were selected on medium containing lactate plus DFG. Strains with mutations in ptsH but not pstI were selected on medium containing fructose plus DFG. Clones isolated from fructose plus DFG semisolid plates and selected for ability to swarm were mutant in either ptsH or ptsG. Mutants of the latter class were defective in enzyme IIB', a membrane component of the glucose transport system. Some pleiotropic properties of one representative ptsG mutant are described.

Familial hypoalphalipoproteinemia (hypoalpha), characterized by a decreased high density lipoprotein level, is associated with an increased incidence of premature cardiovascular disease. Restriction fragment length polymorphism analysis of genomic DNA has detected a polymorphism for the PstI restriction endonuclease near the apoA-I gene, with either a 2.2 or a 3.3 kb fragment. The latter has been previously found to occur with significantly higher frequency in probands of families with familial hypoalpha. ApoA-I was isolated from three unrelated subjects with familial hypoalpha and the 3.3 kb PstI polymorphism of the apoA-I gene, and from normal control subjects. The apoA-I from the hypoalpha subjects was structurally normal as determined by amino acid analysis and by two-dimensional gel electrophoresis. When normal apoA-I and hypoalpha apoA-I were simultaneously injected into either normal controls or hypoalpha subjects, both forms of apoA-I were catabolized at the same rate in the same subject, indicating that the hypoalpha apoA-I is also metabolically normal. Analysis of the kinetics of metabolism of apoA-I in the hypoalpha subjects, compared to the normal controls, revealed that the reduced plasma levels of apoA-I were due to an increased apoA-I fractional catabolic rate, and that the synthetic rate was normal. Based on these results, we conclude that the apoA-I gene in these hypoalpha subjects is normal, and the PstI polymorphism near the apoA-I gene, which is associated with familial hypoalpha, is likely to be a marker for a mutant gene closely linked to, but not in, the apoA-I gene.

Summary Genetic polymorphisms of enzymes involved in carcinogen metabolism have been found to influence susceptibility to cancer. Ethanol-inducible CYP2E1 is an enzyme of major toxicological interest because it metabolizes several drugs, precarcinogens and solvents to reactive metabolites. In the present study, we investigated the cytochrome P450 2E1 genetic polymorphism in renal cell/urothelial cancer patients from two German regions, Jena and Halle, different with respect to their environmental pollution degree in comparison with healthy controls from the same regions. DNA of peripheral white blood cells was isolated both from 224 renal cell/urothelial cancer patients and 304 controls. We focussed on polymorphisms in the promoter region and intron 6 of the CYP2E1 gene. The polymorphisms were identified as RFLP's by amplification of the appropriate DNA fragment and subsequent digestion with the restriction enzymes PstI, RsaI and DraI. In Jena as well as in Halle, the frequency distributions of the PstI/RsaI, DraI and combined DraI + PstI/ RsaI genotypes showed no significant differences between controls and renal cell/urothelial cancer patients. We did not find significant differences between Jena and Halle. 86.2% of all subjects with a homocygote PstI/RsaI genotype also carried a heterocygote DraI genotype, whereas 5.1 % of the subjects with a heterocygote PstI/Rsal genotype also carried a heterozygote DraI genotype. Renal cell cancer as well as urothelial cancer risk was not elevated in patients with heterozygote DraI, PstI/RsaI and combined DraI + PstI/RsaI genotypes (odds ratios slightly insignificantly increased). Interestingly enough, an association between these polymorphisms and renal cell cancer risk was found in the female subgroup but not in the male subgroup. The basis of these sex-specifically increased risks are different frequencies concerning heterozygote and homozygote genotypes in controls and cancer patients. In controls, the heterozygote genotype frequency was lower in females than in males. In renal cell cancer patients, the results were quite the contrary. Summing up, our results demonstrate an lack between CYP2E1 genetic polymorphism and renal cell/urothelial cancer risk.

Six strains containing site-specific endonucleases II were selected from a collection of 45 ice-nucleating bacterial strains isolated from rhizosphere of plants growing in various geographical regions. EndonucleasesPft211I,Psp8I, andPsp23I were isolated and purified from twoPseudomonas sp. strains and aPseudomonas fluorescens strain. Restriction endonucleasesPfl2lI andPsp23I were shown to recognize and cleave the DNA nucleotide sequence 5′-CTGCA↓G-3′. Endonuclease Psp81 recognized and cleaved the DNA nucleotide sequence 5′-G↓GATCC-3′. These endonucleases were found to be true isoschizomers of PstI andBamHI, respectively.

Achondroplasia is the most common genetic form of dwarfism inherited as an autosomal dominant disorder. Individuals affected with achondroplasia have impaired ability to form bone from cartilage (endochondral bone formation). Homozygous achondroplasia is a neonatal lethal condition. The vast majority of patients with achondroplasia have a G-to-A transition at position 1138 of the fibroblast growth factor receptor 3 (FGFR3) cDNA sequence, resulting in the Gly-to-Arg substitution at position 380 of the FGFR3 protein. This mutation has been diagnosed by SfcI digestion of amplified genomic DNA. However, it has also been demonstrated that the SfcI digestion protocol does not consistently distinguish between DNA samples heterozygous and homozygous for the G1138A substitution. This study was designed to improve the molecular diagnosis based on the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques for the FGFR3 G1138A mutation. The newly designed forward primer contains one mismatch (G at position 1136) from the FGFR3 cDNA sequence (A at position 1136), thereby creating a PstI site (CTGCAG at positions 1134 to 1139) in the amplified DNA from alleles containing the G1138A mutation. The PCR-RFLP technique based on the PstI digestion of amplified genomic DNA with a novel forward primer shows 100% accuracy in diagnosis of the G1138A mutation in heterozygous and homozygous individuals. © 2006 Tohoku University Medical Press.

The mitochondrial DNA (mtDNA) of 60 Russians from West Siberia was analyzed with the following restriction enzymes: BamHI, HindIII, PstI, PvuII and SacI that recognize 6 bp. The observed restriction fragment length polymorphisms (morphs) were classified into 13 types of distinct cleavage patterns (mitotypes). The distributions of the mtDNA morphs were compared with those characteristic of some other human populations.

Adrenocorticotrophin (ACTH) is the major regulatory hormone of steroid synthesis and secretion by adrenocortical cells. The actions of ACTH are mediated by its specific membrane receptor (ACTH-R). The human ACTH-R gene was recently cloned, allowing systematic determination of its sequence, expression and function in adrenal tumorigenesis. The presence of oncogenic mutations of the ACTH-R gene in adrenocortical tumors has been reported. Direct sequencing of the entire coding region of the ACTH-R gene of sporadic adrenocortical adenomas and carcinomas did not reveal constitutive activating mutations, indicating that this mechanism is not frequent in human adrenocortical tumorigenesis. Recent studies demonstrated allelic loss of the ACTH-R gene in a subset of sporadic adrenocortical tumors using a PstI polymorphism located in the promoter region of the ACTH-R gene. Loss of heterozygosity of the ACTH-R was analyzed in 20 informative patients with a variety of benign and malignant adrenocortical tumors. Three of them showed loss of heterozygosity of the ACTH-R gene. In addition, Northern blot experiments demonstrated reduced expression of ACTH-R mRNA in these three tumors with loss of heterozygosity, suggesting the functional significance of this finding at the transcriptional level. Deletion of the ACTH-R gene seems to be involved in a subset of human adrenocortical tumors, contributing to cellular dedifferentiation.