The physical map of the chloroplast DNA from Asparagus officinalis L.

The genus Asparagus consists of 100–300 species of both dioecious and hermaphrodite plants. Since there are diploid, tetraploid, and hexaploid plants in this genus, RFLP (restriction fragment length polymorphism) analysis of chloroplast DNA (ctDNA) is suitable for examining the phylogenetic relationships. We have constructed a physical map of the ctDNA of garden asparagus (A. officinalis L. cv Mary Washington 500 W) using five restriction endonucleases, namely, BamHI, PstI, SalI, HindIII, and XhoI. Asparagus ctDNA was digested with restriction enzymes and cloned into plasmid and phage vectors, and a clone bank was constructed that covered 70% of the genome. A physical map was constructed by Southern hybridization of total DNA from asparagus with homologous and heterologous probes. The asparagus ctDNA was about 155 kb long and it contained two inverted repeats (23kb each) separated by a large single-copy region (90kb) and a small single-copy region (19kb). Fifteen genes, encoding photosynthesis-related proteins, rDNAs, and tRNAs, were localized on the physical map of asparagus ctDNA. Comparing the length and the gene order of asparagus ctDNA with that of other plants, we found that asparagus ctDNA was similar to tobacco ctDNA but different from rice ctDNA. The restriction patterns of the ctDNAs from several varieties of A. officinalis and three species of Asparagus were analyzed. The restriction patterns of the varieties of A. officinalis were very similar, but polymorphisms were detected among the three species of Asparagus.     

Clone bank and physical and genetic map of potato chloroplast DNA

Summary Clone banks of PvuII, BamHI and XhoI fragments were generated of the Solanum tuberosum cv Katahdin plastome. These clone banks, in conjunction with molecular hybridization to tobacco ctDNA probes, were used to construct a physical map of potato ctDNA. The potato plastome was found to be a circular molecule of 155–156 Kbp containing two inverted repeat regions of 23–27 Kbp. The arrangement of restriction sites is very similar to that of other Solanaceae plastomes. Heterologous hybridization to known ctDNA encoded gene probes from tobacco allowed us to establish a genetic map of the potato chloroplast genome. The arrangement of these genes on the potato plastome resembles that on most higher plant ctDNAs.     

A cleavage map of the TOL plasmid of Pseudomonas putida mt-2.

Summary A cleavage map of the TOL plasmid pWWO has been determined for the restriction endonucleases HindIII and XhoI. A number of techniques were employed including (i) digestion of purified cleavage products with a second enzyme; (ii) hybridisation of purified XhoI fragments to Southern blots of HindIII digest products and (iii) analysis of a number of deletion mutants.     

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.     

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.     

Restriction map of CELO virus DNA

A restriction map of chicken embryo lethal orphan (CELO) virus DNA was reported with ten restriction endonucleases (XbaI, XhoI, SalI, HindIII, EcoRI, BglI, KpnI, BamHI, PstI and SstI). CELO virus DNA was estimated by comparing CELO virus DNA fragments with marker DNA fragments to have a molecular weight of 29.3·10⁶.     

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.     

Heterogeneity of Maize Cytoplasmic Genomes among Male-Sterile Cytoplasms

Maize mitochondrial and chloroplast DNA's were prepared from normal (fertile) lines or single crosses and from members of the T, C, and S groups of male-sterile cytoplasms. Restriction endonucleases HindIII, BamI, EcoRI, and SalI were used to restrict the DNA, and the resultant fragments were electrophoresed in agarose gels. The results show that the N (fertile), T, C, and S cytoplasms each contained distinct mitochondrial DNA (mtDNA). These distinctive patterns were unaffected by nuclear genotype. No evidence of paternal inheritance of mtDNA was observed. Chloroplast DNA (ctDNA) from the N, C, and T cytoplasms was indistinguishable by HindIII, SalI, or EcoRI endonuclease digestion. The S cytoplasm ctDNA, however, was slightly different from that of other cytoplasms, as indicated by a slight displacement of one band in HindIII digests. The molecular weight of maize ctDNA was estimated to be as high as 88 x 10⁶. Estimates of the minimum molecular weight of maize mtDNA ranged from 116–131 x 10⁶, but the patterns were to complex for an unambiguous determination. Based on HindIII data, a comparison of the molecular weight of mtDNA bands common to the N, T. C, and S cytoplasms suggests that C cytoplasm most closely resembles N cytoplasm. The T and S sources are more divergent from the C and N cytoplasms. These results indicate a possible gradation of relatedness among male-sterile cytoplasms. The marked variation in mtDNA, with apparently less variation in ctDNA, represents circumstantial, but compelling, evidence that mtDNA may be involved in the male sterility and disease susceptibility traits in maize.     

Chloroplast DNA differences between cultivated hop,Humulus lupulus and the related species H. japonicus

Chloroplast DNA (cpDNA) of Humulus Lupulus and H. japonicus was examined by restriction endonuclease analysis with BamHI, BanI, BclI, BstEII, DraI, EcoRI, EcoRV, HindIII, KpnI, PaeR7I, PstI, PvuII, SalI and XhoI. The restriction fragment patterns showed that the cpDNAs shared a large number of restriction sites. However, the chloroplast genomes of the two species could be distinguished by differences in restriction site and restriction fragment patterns in the PstI, PvuII, BclI, EcoRV, DraI and HindIII digests. On the basis of the complexity of restriction enzyme patterns, the enzymes PstI, PvuII, SalI, KpnI and XhoI were selected for mapping the chloroplast genomes. Single and double restriction enzyme digests of cpDNA from the two species were hybridized to cpDNA probes of barley and tobacco. The data obtained from molecular hybridization experiments were used to construct the cleavage site maps. Except for the PstI digest, the arrangement of cpDNA restriction sites was found to be the same for both species. An extra PstI site was present in H. lupulus. Three small insertions/deletions of about 0.8 kbp each were detected in the chloroplast genomes of the two species. Two of these insertions/deletions were present in the large and one in the small singlecopy region of the chloroplast genome. The cpDNA of Humulus was found to be a circular molecule of approximately 148 kbp that contains two inverted repeat regions of 23 kbp each, a small and a large single -copy region of approximately 20 kbp and 81 kbp, respectively. The chloroplast genome of hop has the same physical and structural organization as that found in most angiosperms.     

The molecular cloning of genes specifying some enzymes of the 3,5-xylenol degradative pathway

Enzymes for the degradation of 3,5-xylenol ofPseudomonas putida NCIB9869 are encoded on a transmissible plasmid, pRA500. Genes specifying the inducible synthesis of some enzymes encoded on pRA500, namely, citraconase (G), citramalate coenzyme-A-transferase (H), citramalyl-coenzyme-A lyase (I), maleyl-pyruvate isomerase (J) and fumarylpyruvate hydrolase (K) have been cloned on a 7.9-kbHindIII fragment into the vector pKT231 to give pRA507. Biochemical and restriction analysis of pRA507 and some of its deleted derivatives has enabled preliminary locations to be assigned to the genes encoding these enzymes. The structural genes for enzymes H and I, together with a regulatory gene controlling their expression, are located on a 1.6-kbHindIII/XhoI fragment of pRA507 and the genes for enzymes G, J and K are located within a 4.5-kbXhoI/ClaI fragment of pRA507, which lies immediately adjacent to the former fragment. Biochemical analysis of the strain carrying pRA507, of its plasmid-free derivative and of other plasmid-free derivatives of the wild-typeP. putida has provided indirect evidence for the presence of two isofunctional enzymes at three different steps of the degradative pathway. One set of 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase and maleylpyruvate hydrolase is encoded on pRA500 and these enzymes are inducible only. A second set is encoded on the chromosome and these enzymes are constitutive and not further inducible. The chromosomally located genes appeared to be silent until 3,5-xylenol-negative strains were incubated for prolonged periods with compounds such asm-hydroxybenzoate orp-cresol.     

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).     

Beta chloroplast genomes: analysis of Fraction I protein and chloroplast DNA variation

Summary The interrelationships of Beta chloroplast genomes have been investigated on the basis of the analysis of Fraction I protein and chloroplast (ct) DNA. Three groups of the chloroplast genomes could be demonstrated by the difference in isoelectric points of the large subunit of Fraction I protein. Restriction enzyme analysis revealed inter- and intra-specific variations among the ctDNAs, which enabled us to detect seven distinct ctDNA types. In Vulgares and Corollinae species, the observed differences were physically mapped taking advantage of the restriction fragment map available for sugar beet (B. vulgaris) ctDNA. The DNA variations were found to result either from gains or losses of restriction sites or from small deletions/ insertions, and most of them were located in the large single-copy region of the genome. Moreover, the ctDNAs from Patellares species are more diverged from those of other Beta taxa. Our results also indicate that there is a close correlation between the chloroplast genome diversity and the accepted taxonomic classification of the species included in this survey.     

Cloning and expression in Escherichia coli K-12 of the biosynthetic dehydroquinase function of the arom cluster gene from the eucaryote, Aspergillus nidulans

Summary A 1.35 Md DNA HindIII fragment containing part of the arom gene cluster or cluster gene of Aspergillus nidulans encoding biosynthetic dehydroquinase (5-dehydroquinate hydrolyase) has been cloned in plasmid pBR322 on the basis of functional expression in Escherichia coli. The fungal fragment on pBR322, designated pHK29, complements a corresponding E. coli dehydroquinase structural gene (aroD) mutation. pHK29 contains one BamHI, HpaII, PstI, SmaI, XhoI and surprisingly, one HindIII site since pHK29 hybrid Aspergillus DNA is a HindIII fragment itself. The biosynthetic dehydroquinase activity extracted from E. coli strains, containing pHK29, had properties similar to those of the enzyme activity from Aspergillus. The protein specified by pHK29 appears to be 80 Kd. No increase of dehydroquinase activity was found in polynucleotide phosphorylase deficient strains (pnp) of E. coli.Standard Abbreviations Used SSC Standard saline citrate (3 M Sodium Chloride, 0.15 M Sodium citrate) - EDTA Ethylenediaminetetra acetic acid - DTT Dithiothreitol - PMSF Phenyl methyl sulphonyl fluoride - TEMED N N N N, Tetramethylethylenediamine - Md Megadaltons - Kd Kilodaltons     

A wheat cDNA coding for a thaumatin-like protein reveals a high level of RFLP in wheat

 A cDNA clone that reveals a high level of polymorphism between wheat varieties was isolated from a wheat cDNA library. When hybridized to DraI-, EcoRV- and HindIII digested DNA this clone, gbx3832, enables us to distinguish 42 different patterns among 48 varieties: 37 varieties are clearly identified, the remaining 11 are divided into five groups. Base-sequence analysis of the clone reveals 72–74% sequence identity to mRNAs encoding thaumatin-like proteins from different cereals. Received: 27 January 1997 / Accepted: 18 April 1997     

Polymorphic genotypes of the HRES-1 human endogenous retrovirus locus correlate with systemic lupus erythematosus and autoreactivity

 Antinuclear autoantibodies are a hallmark of systemic lupus erythematosus (SLE). Autoantibodies to HRES-1/p28, a 28 000 M _r nuclear protein, commonly occur in patients with SLE. HRES-1 is a single-copy endogenous retroviral element mapped to human Chromosome 1 at q42. A polymorphic Hin dIII site defines two different allelic forms of the genomic locus. The HRES-1/1 probe [5.5 kilobases (kb)] anneals to three polymorphic fragments and three genotypes can be differentiated: I, 5.5 kb fragment only; II, 3.7 kb and 1.8 kb fragments only; and III, all three polymorphic fragments. By cloning of the HRES-1 locus from homozygous type I and type II human DNA samples, the polymorphic Hin dIII site was identified as a G to C transition at position 653 of the long terminal repeat region. Family studies showed that Hin dIII genotypes of the HRES-1 locus are inherited in a Mendelian pattern. The relative frequency of genotype I with respect to genotype III was 3.1-fold lower in patients with SLE (14 : 40=0.35) in comparison to 100 ethnically matched control donors (47 : 43=1.09;P=0.0084). Frequency of genotype I vs genotype II alleles was lower in SLE (68/52) than in normal donors (137/63;P=0.033), suggesting that a genotype I allele of the HRES-1 locus may be protective against SLE. Western blot seroreactivity with recombinant HRES-1/p28 was noted in 4/14 (29%) of genotype I patients and 13/19 (68%) of genotype III patients (P<0.025). These data raise the possibility that the HRES-1 element or a gene in linkage disequilibrium with this genomic locus may influence autoimmunity in SLE. Received: 20 February 1999 / Revised: 15 April 1999     

A cellulase gene from a new alkalophilic Bacillus sp. (strain N186-1). Its cloning,nucleotide sequence and expression in Escherichia coli

 Several alkalophilic Bacillus spp. strains were selected for their capacity to produce alkaline cellulases. Culture supernatants of these strains showed optimal cellulase activities between pH 8 and 9 and they were stable from pH 6 to pH 12. A cellulase gene (celB1) from the alkalophilic Bacillus sp. strain N186-1 was cloned in Escherichia coli using polymerase chain reaction techniques. The cloned gene was present in a 2.539-bp HindIII fragment and its nucleotide sequence was determined. The coding sequence showed an open-reading frame encoding 389 amino acids. The amino acid sequence, deduced from the nucleotide sequence, permitted us to include it in family 5 (or A) of the glycosyl hydrolases. The complete open-reading frame of celB1 was cloned in the plasmid pET-11d and expressed in E. coli BL21 (DE3), in which a protein of 39 kDa was obtained in the cytoplasm; however, no endoglucanase activity was detected. A second construction in pET-12a allowed the production of a 39-kDa protein located in the periplasmic space of E. coli that had endoglucanase activity. The protein produced has optimal activity at pH 7 and 50°C and it retains more than 70% of its activity after incubation for 1 h at pH 12. Received: 27 December 1995/Received revision: 14 March 1996/Accepted: 25 March 1996     

A plasmid cloning vector for KpnI-cleaved DNA

A plasmid cloning vector containing a single site for KpnI has been generated by insertion of a 3.5-kb EcoRI/HindIII fragment of pCR1 into the EcoRI/HindIII sites of pBR322. KpnI cleavage yields 3′ rather than 5′ “sticky ends” which allows reconstitution of the recognition site after cloning by a homopolymer joining procedure. This is an advantage shared with only one or two other commercially available restriction enzymes.     

Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid

Cleavage of the group-A streptococcal macrolide, lincosamide, and streptogramin B (MLS) resistance plasmid pSM19035 yields 2 fragments [13 and 4 megadaltons (MD)] with EcoRI, and 15 fragments with HindIII, 12 of which are 6 pairs of identical fragments derived from the inverted repeats that comprise about 80% of the pSM19035 genome. The large EcoRI fragment was isolated, ligated, and used to transform the Challis strain of Streptococcus sanguis to erythromycin resistance. Plasmids (pDB101, pDB102, and pDB103) isolated from three different transformants had lower molecular masses than the original large EcoRI fragment. HindIII digestion of these molecules and subsequent analysis of fragment radioactivity distributions indicated the loss of plasmid segments of various sizes. The deletions, all of which occurred in the palindrome, did not affect the level and the inducible nature of pSM19035-determined antibiotic resistance. Only pDB101 retained the unique EcoRI cleavage site. The results of this analysis allowed the construction of an EcoRI and HindIII cleavage-site map of pSM19035 and promise to simplify future studies of genetic functions specified by streptococcal MLS resistance plasmids.     

Multiple physical differences in the genome structure of functionally related bacteriophages P1 and P7

Summary 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.Abbreviations Cm chloramphenicol - Ap ampicillin - bp base pairs - kb kilo-base pairs     

Analysis of oral infection and helicase gene of the nucleopolyhedroviruses isolated from Philosamia cynthia ricini and Antheraea pernyi

Philosamia cynthia ricini is an important commercial silkworm in Asia. In this report, a nucleopolyhedrovirus isolated from P. cynthia ricini (PhcyNPV) larva was purified and compared with Antheraea pernyi nucleopolyhedrovirus (AnpeNPV), a pathogen of A. pernyi, another commercial silkworm in China. The two viruses had similar polyhedral morphology and shared high sequence homologue of viral fragments including the p143 gene. However, the restriction fragments, digested with SalI, XhoI, HindIII and PstI, respectively, were different. The cross-infectivity of the two viruses was also tested. AnpeNPV caused 57% mortality in larvae of P. cynthia rici, whereas PcrNPV did not kill larvae of A. pernyi. Results indicated that PhcyNPV and AnpeNPV had closed relatedness, and that PhcyNPV might be a variant of AnpeNPV.