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.     

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.     

Physical map of DNA from a new cauliflower mosaic virus strain

The restriction enzymes AluI, BamHI, BglII, EcoRI, HindIII, and SalI have been used to characterize and map a new cauliflower mosaic virus strain (Cabb-S). These fragments have been ordered by examining their overlapping regions after double enzymatic digestion. The single SalI cleavage site was chosen as the point of origin. We compare this strain with those already described.     

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.     

A physical map of plasmid pDU1 from the cyanobacterium Nostoc PCC 7524

Nostoc 7524 contains three different plasmids of molecular weight, 4, 8, and 28 Mdal. The smallest plasmid, designated pDU1, because of its size and ease of isolation, may prove to be useful as a cloning vector. Plasmid pDU1 was incubated separately with 26 different restriction enzymes and only 8 of the enzymes tested cut pDU1. A composite restriction enzyme map consisting of a total of 17 restriction sites was constructed for BglI, HindIII, HpaI, and XbaI. The sites of restriction enzyme cleavage were determined by single, double, and partial digests of plasmid DNA or redigestion of isolated restriction fragments. All the restriction sites were aligned relative to the single BglI site. This is the first restriction enzyme map of a plasmid from a filamentous cyanobacterium.     

A restriction enzyme map of R-plasmid RP1

The relative positions of the sites on RP1 of the following restriction enzymes were mapped: EcoRl, BamH1, HindIII, BglII, SmaI, andPstI.     

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.     

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.     

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.     

A plasmid cloning vector for Kpnl-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.     

Restriction site mapping of adenovirus type 8 genome types

The DNA of 60 adenovirus type 8 (AV8) isolates (collected during the period 1961 to 1982, mostly in Western Germany) was analysed by 6 endonucleases and revealed 6 different genome types, thus implying that the variability of AV8 is relatively low. It was found that 45 isolates belonged to the genome type D1. Restriction site maps of a prototype D1 and of all deviating restriction variants were elaborated for enzymes BamHI, BglII, Hind-III and SalI.     

A HindIII/BglII dystrophin gene polymorphism in the African-American population

Summary We describe a common dystrophin gene polymorphism in the black population that alters both HindIII and BglIII restriction sites.     

Cloning of the yeast ribosomal DNA repeat unit in SstI and HindIII lambda vectors using genetic and physical size selections.

The size of DNA fragments complementary to ribosomal RNA was determined in SstI and HindIII restriction spectra from totally and partially cleaved yeast (Saccharomyces cerevisiae) DNA. The results indicated that the yeast ribosomal RNA gene cluster consists of 9000 base-pair long tandemly repeated units. Three different repeating units, which are overlapping with respect to their sequences, were cloned as SstI and HindIII fragments with λ vectors. The isolation of these clones was facilitated by genetic or physical preselection for those recombinant phage which contained DNA inserts in the expected size range. Both preselection methods gave about a 30-fold purification with respect to the λ-rDNA clones. A heteroduplex analysis of the clones obtained with a three-component HindIII vector showed that the center part of the λ genome carrying λ recombination and regulation genes (57 to 77% λ) can become inverted without apparent decrease of growth capacities.     

Restriction site periodicities in highly repetitive DNA of primates.

Highly repeated DNA sequences from three Old World primate groups have been compared, using restriction endonucleases. Baboons, macaques and mangabeys share a 340⁴ base-pair, tandemly repeated DNA that is cut once by EndoR · BamHI. The several species of guenons, including the African green monkey, possess a related 170 base-pair, tandemly organized sequence distinguished by the feature of being cut once by EndoR · HindIII, EndoR · MboII or EndoR · HphI. The tandemly repeated DNA of the colobus monkey is based on a monomer length of 680 base-pairs, being cut once by EndoR · BamI or EndoR · EcoRI. Thus, all three highly repeated DNAs have a monomer length of 170n base-pairs, where n = 1, 2 or 4. The 340 and 680 base-pair repeated DNAs contain an internal 170 base-pair periodicity with respect especially to the EndoR · HindIII cleavage site, but with respect also to several other enzymes that characterize each repeated sequence. The 170 base-pair length is called the fundamental unit.The three repeated DNAs are more conserved in the region around the HindIII site and are more divergent elsewhere in the sequence. All seven 170 base-pair fundamental units were related to one another, judging from the overall similarities of the maps of restriction endonuclease cleavage sites. The highly repeated DNAs from baboons and guenons are related enough to cross-hybridize at relaxed criteria (60 °C in 0.12 m-Na⁺) but neither hybridizes to repeated colobus DNA under this condition.The results show that highly repeated sequences in primates form a common library descended from a single ancestral sequence, with 170 base-pairs making up the fundamental unit of library members. Occasionally, a member of the library is amplified, creating a newly amplified family. In Old World monkeys the most recent amplification just preceded active speciation.     

Characterization of the ColE1-Km plasmids pCR1 and pCR11 by electron microscope and restriction endonuclease mapping.

The ColE1-Km plasmids pCR1 and pCR11 have been characterized by electron microscope techniques. Their sizes have been determined to be 13.1 and 9.2 kb, respectively, and heteroduplex studies show that the plasmids differ in the presence of a 3.9 kb deletion in the ColE1 region of pCR11. Both contain a nontandem inverted duplication of a 1.06 kb sequence. The single HindIII site, 3.5 kb from the EcoR1 site, lies in the 0.97 kb of DNA between the inverted repeat sequences. Since DNA insertions at the HindIII site destroy kanamycin resistance, it can be concluded that the kanamycin phosphotransferase gene is contained in this region. Electron microscopy of self-annealed plasmids treated with restriction endonucleases shows that each inverted duplication sequence contains one HindII site and at least two SmaI sites.     

Identification of <Emphasis Type="Italic">Ganoderma</Emphasis>, the causal agent of basal stem rot disease in oil palm using a molecular method

From comparison of the alignments of the internally transcribed spacers (ITS) of ribosomal DNA from Ganoderma associated with oil palm basal stem rot (BSR) and other Ganoderma species, two specific primer pairs were selected to provide a specific DNA amplification of pathogenic Ganoderma in oil palm. Each primer pair produced a single PCR product of about 450 bp (for primer pair IT1–IT2) and 334 bp (for primer pair IT1–IT3) when oil palm Ganoderma DNA was used. No PCR amplification product was observed when other Ganoderma species DNA was used in PCR amplification with these primer pairs. Three specific restriction enzyme sites were identified in the ITS and intergenic spacer (IGS1) regions. The restriction enzymes MluI, SacI and HinfI were used to digest the ITS-PCR product and restriction enzymes TfiI, ScaI and HincII were used to digest the IGS1-PCR product. Of the three restriction enzymes used in each rDNA region, MluI specifically digested the ITS regions, and TfiI specifically digested the IGS1 region of oil palm Ganoderma. Analysis of the published ITS nucleotide sequences of 31 Ganoderma species showed that the MluI restriction site was not present in other Ganoderma species. The use of both specific primers and restriction enzyme analysis can be applied as a standard protocol to identify pathogenic Ganoderma in oil palm. In this study, the use of specific primers and PCR-RFLP analyses of the rDNA gave consistent results for the characterisation of pathogenic Ganoderma, and indicated that Ganoderma strains associated with BSR disease in oil palms belong to a single species.     

PCR-Restriction Fragment Length Polymorphism Analysis of the Phospholipase B (PLB1) Gene for Subtyping of Cryptococcus neoformans Isolates

Cryptococcus neoformans is a pathogenic yeast that is currently divided into three varieties, five serotypes, and eight molecular types. The following report describes the use of PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) as a simple tool to differentiate between C. neoformans subgroups. A PLB1 fragment, 1,970 bp, was amplified and digested with either AvaI or HindIII. Both sets of profiles grouped the isolates into their respective varieties, but only the AvaI profiles allowed for the identification of the eight molecular types via the corresponding RFLP profiles A1 to A8. Digestion of the same fragments with HindIII resulted in RFLP profiles H1 to H5, which distinguished only between serotype A, AD, D, and B/C. Neither enzyme distinguished serotype B from serotype C. The serotype AD profile was a composite of the serotype A and D profiles. Further investigation showed that the serotype AD isolates used in this study are heterozygous, with one allele of PLB1 originating from a serotype A parent and the other from a serotype D parent.     

Length polymorphisms, restriction site variation, and maternal inheritance of mitochondrial DNA of Drosophila melanogaster

We have studied the mitochondrial DNA in three wild type laboratory strains of Drosophila melanogaster, ry⁺⁵ and two Oregon R-substrains, called here R and E. Lengths of the restriction bands for EcoRI, BglII, HpaII, MspI, HaeIII, and HindIII were compared. The number of restriction sites was identical in all strains, with the exception of an extra HaeIII site in ry⁺⁵. Careful comparison of restriction fragment lengths showed that bands containing the AT-rich region were different in length among all strains. The laboratory strains, ry⁺⁵, proved to be a mixture of strains carrying different mtDNAs; these separated into substrains G1 and G2 in the progeny of single pair matings. Adult progeny of reciprocal crosses of G1 and R were analyzed by HaeIII restriction digestion. The results demonstrated maternal inheritance for both the extra restriction site and band containing the AT-rich region.     

Physical and functional map of the hemolytic plasmid pSU316

A restriction endonuclease analysis of the hemolytic plasmid pSU316 has allowed location of the cleavage sites for the endonucleases BamHI, XbaI, KpnI, BglII, SalGI, EcoRI, and HindIII. Hybridization experiments between pSU316 and pED100 have shown that the tra region of pSU316 lies in a segment comprising part of SalGI fragments S-1 and S-3 and the entire fragment S-4. The positions of other plasmid coded functions, namely the replication functions and α-hemolysin production, have been determined in the physical map.     

Evolutionary relationships between laboratory mice and subspecies ofMus musculus based on the restriction fragment length variants of the chymotrypsin gene at thePrt-2 locus

Restriction endonuclease fragment length variants in mice were compared by Southern blot analysis using the cDNA probe pcXP33 for the chymotrypsin gene. The variants were detected in the restriction patterns generated by fragments from digestions withBglII,EcoRI,HindIII,Pstl,SacI, andXbaI. The set of protein phenotypes and the restriction patterns of chymotrypsin gene were examined in many laboratory strains and wild subspecies. Most laboratory strains (26 strains) are grouped into a set defined as Set 1, but only a few laboratory strains (AU/SsJ and five BALB/c sublines) are classified as belonging to Set 2. Of wild subspecies, only BRV-MPL (M. brevirostris) can be placed in Set 1, while DOM-BLG and SK/Cam (M. domesticus) belong in Set 2. The assignment of an appropriate set defined by the characteristics of the chymotrypsin gene has also been investigated inM. musculus, two Chinese subspecies,M. yamashinai, M. molossinus, andM. castaneus, and the evolutionary relationship between laboratory mice and various subspecies ofMus has been examined.