Somatic cell mapping of the bovine prion protein gene and restriction fragment length polymorphism studies in cattle and sheep

Brains affected by the progressive neurological disease bovine spongiform encephalopathy (BSE) contain scrapie-associated fibrils and the protease-resistant isoform of prion protein. The gene encoding the normal host prion protein (PRNP) has been mapped to human chromosome 20 and mouse chromosome 2 with the hamster cDNA probe pEA974. Using this probe and a panel of bovine-rodent hybrid somatic cells, we have mapped PRNP to bovine syntenic group U11 (100% concordancy). PRNP restriction fragment length polymorphisms (RFLPs) were detected with five of six enzymes (BglII, EcoRI, HindIII, MspI and TaqI) in sheep, in contrast to one of 16 enzymes (HincII) in cattle. Codominant segregation of the bovine HincII RFLP was demonstrated in six backcross pedigrees. While PRNP RFLPs are tightly linked to scrapie incubation period, and consequently susceptibility or resistance to disease in rodents and sheep, the relationship between the PRNP RFLPs and BSE incubation period has not been determined.     

Organization of the interferon-inducible 2′,5′-oligoadenylate-dependent ribonuclease L (RNase L) gene of mouse

Interferons (IFNs) induce a 2′,5′-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) following virus-infection of mammalian cells. RNase L degrades both viral and cellular RNAs and restricts virus-proliferation. We have studied organization of RNase L gene in genomic DNA from the mouse liver by Southern blot analysis. Several BamHI, BglII, EcoRI, HincII, HindIII, NcoI, PstI, SacI, and XbaI restriction fragments hybridized to ³²P-labeled mouse RNase L cDNA and the 5′-proximal exon probes. Mouse RNase L gene exists as a single copy (>16 kb DNA) gene. A 5 kb HindIII and a 2.5 kb EcoRI DNA were detected as 5′-upstream DNA of the gene which may contain mouse RNase L promoter. Our results will help studying mouse RNase L gene promoter in further details.     

Study of restriction fragment length polymorphisms at the human phenylalamine hydroxylase locus and evaluation of its potential application in prenatal diagnosis of phenylketonuria in Chinese

Summary Using a human phenylalanine hydroxylase cDNA probe, the restriction fragment length polymorphisms at the human phenylalanine hydroxylase locus have been determined with the restriction enzymes BglII, PvuII, EcoRI+BamHI, MspI, XmnI, HindIII and EcoRV. The frequency of the observed heterozygosity of the restriction site polymorphisms at this locus in a Chinese population is approximately 54%, which is significantly lower than that in Caucasians. No DNA rearrangement or deletion of the phenylalanine hydroxylase locus was detected among mutant phenylalanine hydroxylase genes in seven Chinese classical phenylketonuria (PKU) families. Haplotype analysis of these seven families revealed that the mutant alleles belonged to five different haplotypes, i.e. haplotype 4, 11 and three unreported haplotypes. The majority of normal and mutant phenylalanine hydroxylase genes are confined to hyplotype 4. These results indicate that approximately 42% of Chinese PKU families are informative for prenatal diagnosis of PKU when eight restriction sites linked to the phenylalanine hydroxylase locus are examined.     

Restriction cleavage maps of coliphages 186 and P2

Summary The restriction enzymes BamHI, BglII, 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, BglII-1, EcoRI-3, HindIII-2, PstI-22, XbaI-0 and XhoI-1; b) P2, BamHI-3, BglII-2 EcoRI-3, HindIII-0, PstI-3, XbaI-1 and XhoI-0. 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.     

A restriction enzyme cleavage map of the histidine utilization (hut) genes of Klebsiella aerogenes and deletions lacking regions of hut DNA

Summary 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.     

Physical mapping of BglII, BamHI, EcoRI, HindIII and PstI Restriction fragments of bacteriophage P1 DNA

Summary 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.     

Molecular cloning and physical map of bacteriophage PM2 DNA

The entire genome and the DNA fragments of the lipid-containing bacteriophage pM2 were cloned in the pBR322 plasmid vector. A physical map including the sites for the following restriction enzymes was obtained: HpaII, HaeIII, TthI, Sau96I, AvaII, PstI, BstNI, AccI, HincII, HpaI and HindIII. No restriction sites on PM2 DNA were found for BalI, BamHI, BclI, BglI, BglII, BstEII, KpnI, PvuII, SacI, SalI, Sau3A, XbaI and XhoI.     

MspI andHindIII restriction fragment length polymorphisms at the human Na,K-ATPase β-subunit (ATP1B) gene locus

The authors report on four restriction fragment length polymorphisms detected at the ATP1B gene locus with the restriction endonucleasesHindIII andMspI.     

Analysis of a polymorphic region of the Euglena gracilis chloroplast genome

The circular chloroplast genome of Euglena gracilis is known to carry a single region of variable length (polymorphic region) located on BglII·Z fragments (6.1–6.9 kbp). We now have cloned a BglII·Z fragment (6.35 kbp) using as vector a modified pBR322. The cloned BglIl·Z fragment is split by HindIII into fragments of 1.0, 2.6 and 2.75 kbp and by HaeIII into fragments of 0.05, 1.35 and 4.95 kbp, respectively. The zone of variable length on this BglIl·Z fragment can be confined to a BglII-HindIII fragment of 2.6 kbp which is next to the previously mapped BglIl·G₁ (4.7 kbp) and about 4.5 kbp away from the 5′ end of the ‘extra’ 16 S rRNA gene. Two HindIII fragments from the polymorphic region of 5.9 and 6.1 kbp, respectively, were cloned and used in electron microscopic studies. Heteroduplexes formed between the two cloned HindIII fragments show the expected size difference of about 200 bp. Single strand reannealing experiments allow us to place the polymorphic region between two previously mapped short inverted repeats and partial DNA melting experiments indicate that the polymorphic region is very rich in dA-dT.     

Permutation of the DNA in small-headed virions of coliphage P1

Summary 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.     

Physical characterization of the plasmid pON5300

Summary The antibiotic resistance plasmid Rldrd19Km-which has spontaneously lost its kanamycin resistance marker, and its derivative pON5300, were analysed using the restriction endonucleases SalI, BamHI, HindIII and EcoRI. The fragment patterns were compared with that of the Rldrd19 and the fragments responsible for kanamycin resistance were found to be missing in Rldrd19Km ^– and pON5300. In these plasmids a 7 Mg/mol EcoRI fragment was observed instead of the D (6.3 Mg/mol) fragment of Rldrd19. Further a new 6 Mg/mol EcoRI restriction fragment was observed in pON5300. Using double digestions it was shown that the new fragment does not carry restriction sites for HindIII, BamHI and SalI endonucleases. The non-homology of the analysed plasmid was proved electron microscopically by heteroduplex techniques. The possibility of amplification in the regulatory region for the expression of R-determinants in pON5300 is discussed.     

DNA polymorphisms in the ε- and γ-globin gene regions in asian macaques

DNA polymorphisms in the ε- and γ-globin gene regions in nine Asian macaques(Macaca fuscata, M. mulatta, M. nemestrina, M. cyclopis, M. fascicularis, M. arctoides, M. radiata, M. maura, andM. assamensis) were examined using several different restriction endonucleases and the human ε and γIVS2 probes.M. fuscata andM. mulatta had highly polymorphic sites(BglII andHincII, respectively) in the ε-globin gene region. TheBamHI site in the γ-globin gene region was highly polymorphic inM. mulatta, while theBglII andHindIII sites in the γ-globin gene region were highly polymorphic inM. fuscata andM. mulatta. The presence of the γ-globin gene triplication was frequently observed inM. fuscata. The gain or loss of an additionalBglII site between two γ-globin genes in Asian macaques seems to be very meaningful for discussing genetic relationships among them. This study was supported in part by the Cooperation Research Program of the Primate Research Institute, Kyoto University.     

Analysis of rotifer ribosomal gene structure using the polymerase chain reaction (PCR)

We have used the polymerase chain reaction (PCR) to selectively amplify 18S ribosomal genes in rotifer taxa from major planktonic clades. In each case, we obtained an amplified product of between 1.8 and 2.0 kilobase pairs. We analyzed the PCR products using 6- and 4-base cutting restriction enzymes, comparing fragment mobilities. For example, Brachionus plicatilis (BSL strain) 18S genes have no restriction sites for Hind III or Bam HI and only a single site for Eco RI (all 6-base cutters). The 4-base cutter Msp I, on the other hand, has at least 4 enzymatic sites, producing fragments between approximately 110 and 460 base pairs in length. Results of this type can be used to differentiate among species and species groups within the Rotifera and can be used as the basis for construction of a broad molecular phylogeny of the group.     

Characterization of plQ5 plasmid originating fromKlebsiella pneumoniae

plQ5 plasmid consists of a group of genes specifying resistance to ampicillin, chloramphenicol, carbencillin, kanamycin and trimethoprim-sulphamethoxazole. It is isolated inKlebslella pneumoniae ZD532, is about 26.8 Kb and is freely transmissible to various bacterial species of Gram-negative bacteria. Physical characterization revealed that plQ5 plasmid has a single site forHindill,BamHI,EcoRI and two sites forBglII restriction enzyme.

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Résumé Le blasmide plQ5 consiste en un groupe de genes condant pour la résistance à l'ampicilline, au chloramphénicol, à la carbencilline, à la kanamycine et au triméthoprimsulphamethoxazole. Il est isolé dansKlebsiella pneumoniae ZD532; sa longueur est d'environ 26.8 kb et il est librement transmissible à diverses espèces bactériennes Gram-négatives. La caractérisation physique révèle que le plasmide plQ5 a un site unique pour les enzymes de restrictionHindill,BamHI,EcoRI et deux sites pourBglII.

     

Genetic analysis of the phosphinothricin-tripeptide biosynthetic pathway of Streptomyces viridochromogenes Tü494

Summary Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricyl-alanyl-alanine (Ptt). Ptt-non-producing mutants were isolated following N-methyl-N-nitro-N-nitrosoguanidine (NTG) or UV light treatment of spore suspensions. In co-synthesis and bioconversion experiments the mutational blocks in the biosynthetic pathway were localized. The mutant NTG1 was analysed in detail. This mutant acts as a secretor for all other mutants. From bioconversion experiments with presumptive precursors circumstantial evidence was obtained that NTG1 is mutated in a gene involved in the alanylation of N-acetyl-demethyl-phosphinothricin. Using a cosmid gene library the DNA region complementing the defective gene of mutant NTG1 was isolated on a 4-kb BamHI fragment. Subcloning experiments showed that a 3-kb BglII/BamHI fragment is sufficient for complementation of mutant NTG1.Formerly Susanne MüllerOffprint requests to: W. Wohlleben     

Linkage of the DNA-segment D7S13 (pB79a) with the cystic fibrosis locus

Summary A map distance of 2.9 cM between D7S13 (pB79a) and the cystic fibrosis (CF) locus was obtained from the analysis of 13 informative families with a history of CF. This result is based solely on the HindIII restriction fragment length polymorphism (HindIII-RFLP) at D7S13, since the interpretation of the Msp1-RFLP at this locus was found to be unreliable.     

Restriction enzyme map for streptomycete plasmid pUC3

A restriction enzyme map for the streptomycete plasmid pUC3 was constructed for the enzymes XhoI, EcoRI, HindIII, PstI, BamH-I, and BglII. The plasmid was isolated from Streptomyces sp. 3022a which produces chloramphenicol and has been referred to as S. venezuelae (Bewick et al., 1976 and Bewick and Williams, 1977, Microbios, 19, 27–35).     

Overlapping of the coding regions for alpha and gamma components of penicillin-binding protein 1 b in Escherichia coli

Summary The mode of biosynthesis of penicillin-binding protein(PBP)-1 b in Escherichia coli was investigated by use of the plasmid carrying the ponB(PBP-1 b) gene region. Analyses of the products synthesized in minicells and in vitro showed that PBP-1 b was synthesized as two molecular species corresponding to the and components of PBP-1 b. The coding regions for the and components were located within the ca. 3.7 kb MluI-HincII fragment and transcribed in the direction from the HincII to the MluI site. The capacity for producing the component was abolished by a deletion extending to the MluI site ca. 0.7 kb inward from the HincII end of the ca. 3.7 kb fragment; the remaining 3.0 kb region with the MluI site at both ends directed the production of the component alone. The production of the component was enough to correct all the known defects caused by a ponB mutation. In addition to these results, the analyses for cross-reacting materials produced in correspondence to the various deletions indicated that the coding regions for the and components overlapped and that the N-terminal portion was responsible for the difference between the two components. The distal region about 0.7 kb long inward from the MluI end of the MluI-HincII fragment was dispensable for producing the functional PBP-1 b, although the PBP-1 b produced was curtailed. By a larger distal deletion reaching almost to the middle of the MluI-HincII fragment, the polypeptide produced for PBP-1 b lost the ability to bind penicillin and still retained a low but significant activity for glycan synthesis. We suggest, therefore, that the polypeptide portion required for transglycosylase activity resides on the N-terminal half of PBP-1 b, followed by the middle portion necessary for penicillin-binding and the C-terminal part dispensable for the function of PBP-1 b.     

Characterization of a genomic clone of Erwinia carotovora subsp. carotovora TG3 encoding a pectolytic enzyme of apparent molecular weight 78 kDa

Summary A clone containing the gene encoding a pectolytic enzyme of Erwinia carotovora subsp. carotovora was selected as the one that showed maceration on a solid medium containing sodium polypectate. The gene was located on a 3.2-kb DNA fragment flanked by a BglII site and a Hin-dIII site. Via mini-Mudlac mutagenesis, a possible promoter site was located within the gene between the BglII site and the EcoRI site. The mRNA transcribed from the promoter was directed from the BglII site toward the EcoRI site, determined from the orientation of the inserted mini-Mudlac. The probable gene product was identified as a 78 kDa protein. The enzyme activity of the Escherichia coli clone was detected mainly in the periplasmic space. Potato tuber slices were not macerated by the E. coli clone and synthesis of the enzyme in E. coli was not regulated by the enzyme substrate, sodium polypectate.