Leptospira genomes are modified at 5'-GTAC.

Genomic DNAs of 14 strains from seven species of the spirochete Leptospira were resistant to cleavage by the restriction endonuclease RsaI (5'-GTAC). A modified base comigrating with m4C was detected by chromatography. Genomic DNAs from other spirochetes, Borrelia group VS461, and Serpulina strains were not resistant to RsaI digestion. Modification at 5'-GTAm4C may occur in most or all strains of all species of Leptospira but not in all genera of spirochetes. Genus-wide DNA modification has rarely been observed in bacteria.     

Studies on four restriction fragment length polymorphisms of the type I collagen genes in two Italian populations

Type I collagen, the most abundant of the collagen protein family, is encoded by two genes, COL1A1 and COL1A2. Two random population samples, one from central Italy and one from southern Italy, were studied for 1 restriction fragment length polymorphism (RFLP) of COL1A1 (RsaI) and 3 RFLPs of COL1A2 (EcoRI, RsaI and MspI). A considerable heterogeneity for COL1A1/RsaI was found not only between Italians and English but even among Italians. The potential usefulness of these RFLPs and haplotypes as anthropogenetic markers, particularly in distinguishing Caucasoids from Negroids, has been discussed.     

Effect of nucleotide analogs on the cleavage of DNA by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI

The cleavage of specific DNA sequences by the restriction endonucleases AluI, DdeI, HinfI, RsaI, and TaqI has been studied by monitoring the effect of various nucleotide modifications on the rate of DNA digestion. Bacteriophage fd DNA was completely substituted in one strand with a single nucleotide analog, using an in vitro primed DNA synthesis reaction on a single-stranded viral DNA template. Twelve deoxynucleotide analogs were incorporated into these DNA substrates: 2-aminopurine, 2,6-diaminopurine, deoxytubercidin, deoxyuridine, 5-bromodeoxyuridine, 5-allylamine deoxyuridine, 5-biotinyl deoxyuridine, deoxypseudouridine, deoxyinosine, 8-azadeoxyguanosine, 5-iododeoxycytidine, and 5-bromodeoxycytidine. The restriction enzymes tested varied considerably in their ability to digest hemi-substituted DNAs containing these modified nucleotides. Structural alterations in the base pairs immediately adjacent to the phosphodiester bonds cleaved by the enzyme reduced the rate of enzyme activity most dramatically, and in most cases more than a single determinant on each base pair altered activity. Interactions with nucleotides outside the recognition site seem to have little importance in the binding or catalytic activity of these enzymes.     

Multiple cellular factors bind to cis-regulatory elements found inboard of the 5' palindrome of minute virus of mice.

Previous genetic analysis of the DNA replication of minute virus of mice (MVM) minigenomes suggested that specific elements, A (nucleotides [nt] 4489 to 4636) and B (nt 4636 to 4695), found inboard of the 5' palindrome are required for efficient MVM DNA replication (P. Tam and C. R. Astell, Virology 193:812-824, 1993). In this report, we show that two MVM RsaI restriction fragments (RsaI A [nt 4431 to 4579] and RsaI B [nt 4579 to 4662]) are able to activate DNA replication of an MVM minigenome containing deletions of both elements A and B. We also show that sequences inboard of the right palindrome are able to activate replication of minigenomes containing two left termini. In order to investigate the importance of the RsaI fragments, we demonstrate the presence of a number of sequence-specific DNA-protein interactions by electrophoretic mobility shift assays. After partial fractionation of A9 nuclear extracts, DNase I footprinting analysis was used to determine the binding sites for MVM replication factor (MRF) B5. MRF B5 protects two distinct regions (sites I and II) of the RsaI B probe from DNase I digestion. Competition f electrophoretic mobility shift assays with synthetic oligonucleotides corresponding to sites I and II suggest that MRF B5 is composed of two factors, MRF B3 and MRF B4, which bind DNA independently in a sequence-specific manner. It may be possible that these replication factors are proteins which are able to transactivate MVM DNA replication and hence are accessory replication factors.     

Detection of a novel DNA polymorphism in the beta-globin gene cluster

Analysis of DNA from the beta-globin gene cluster in an Albanian family identified a novel RsaI site approximately 550 base pairs 5' to the beta-globin gene. In this family, two chromosomes carrying otherwise identical beta-globin haplotypes were found to differ at the RsaI site. Population screening demonstrated the presence and absence of the site in DNA from individuals of northern European, Mediterranean, Middle Eastern, Southeast Asian, African, and Asian Indian descent, indicating that this site is a DNA polymorphism common in many ethnic groups. The polymorphism is also present in DNA from individuals carrying different beta-globin alleles. Additional nucleotide sequence changes identified in an RsaI (+) genomic clone in the region immediately 3' to the RsaI site suggest a mechanism for the randomization of the site with respect to haplotype.     

DNA sequence selectivities in the covalent bonding of antibiotic saframycins Mx1, Mx3, A, and S deduced from MPE.Fe(II) footprinting and exonuclease III stop assays.

DNA sequence selectivities in the covalent binding of the antitumor antibiotic saframycins Mx1, Mx3, A, and S have been determined by complementary strand MPE.Fe(II) footprinting and exonuclease III stop assays on two different 545 and 135 base pair long HindIII/RsaI restriction fragments of pBR322 DNA. Saframycins Mx1, Mx3, A, and S recognize primarily 5'-GGG sequences. All four antibiotics also recognize 5'-GGPy sequences, however a cytosine is preferred over a thymine at the 3'-end of this recognition site in all cases. Saframycins Mx1, Mx3 and S, which possess the OH leaving group, also recognize the 5'-CCG sequence, in contrast to saframycin A, which contains the CN leaving group. In contrast, the OH-containing saframycins also recognize the 5'-CTA sequence. Saframycins Mx2, B and C, which lack the critical CN or OH leaving group, do not show any footprints on the restriction fragments examined in this study. The measured binding site size for all four antibiotics is three base pairs. The exonuclease III stop assay independently confirmed the formation of a covalent bond and the strong preference of the antibiotics for 5'-GGG and 5'-GCC sequences. The latter enzyme assay also suggests that the 5'-terminal or central G of the triad binding site is the base to which reversible covalent attachment of the antibiotic takes place.     

Detection of a high frequency RsaI polymorphism in the human pro alpha 2(I) collagen gene which is linked to an autosomal dominant form of osteogenesis imperfecta.

Screening of the pro alpha 2(I) collagen genes of Southern African populations for restriction fragment length polymorphisms (RFLPs) has revealed a locus polymorphic for the restriction enzyme RsaI. The frequency of the RFLP was 0.38 in Afrikaners, but much lower in indigenous Southern African populations, which suggests that it is of European origin. The polymorphism was used to study 19 affected and non-affected individuals in a four generation family with the autosomal dominant disorder, osteogenesis imperfecta (OI) type I. Co-inheritance of the loss of the RsaI site and the OI phenotype was observed with a lod score of 3.91 at a recombination fraction (theta) of zero, indicating strong linkage. This suggests that the defect in this family is caused by a structural mutation within or close to the pro alpha 2(I) collagen gene. The use of this high frequency RFLP together with other recently described polymorphisms at this locus will facilitate the analysis of the role of this gene in OI and other inherited disorders of connective tissue.     

PCR-based identification of individuals of Schistosoma japonicum representing different subpopulations using a genetic marker in mitochondrial DNA.

A mitochondrial NADH dehydrogenase I gene fragment (NDI) was sequenced for three laboratory maintained isolates of Schistosoma japonicum. Comparison of sequences representing the isolates (originally obtained from the Anhui and Zhejiang provinces of the People's Republic of China, and from the Philippines) revealed inter-isolate sequence variations of 0.2-0.6% and no intra-isolate variation was found. The sequences also indicated that while the amplification products of the Zhejiang and Philippine isolates contained a recognition site for the endonuclease RsaI, there was no such site in the Anhui isolate. This was tested by digesting amplification products from a number of individual worms with RsaI. Then an infection experiment was designed to test the value of this genetic marker for studies of the population biology of S. japonicum in the final host. For this, the two Chinese isolates were used. Three groups of mice (A-C) were exposed firstly to a primary infection and then challenge-infected at weeks 4 and 7 of the experiment. In group A the first infection was done with the Anhui isolate, and the two others with the Zhejiang isolate, thereby providing a specific, detectable cohort. In groups B and C the Anhui isolate was used for the second and third infection. All mice were perfused 5 weeks after the last challenge infection, and the NDI was subsequently amplified from DNA of the perfused worms and digested with RsaI. The digestion revealed that while infection groups A and B contained mixed populations of the Anhui and Zhejiang isolates, only Zhejiang worms were present in group C. We concluded that the absence/presence of the RsaI site in the NDI provides a useful marker for the delineation of cohorts of S. japonicum.     

DNA sequence recognition by under-methylated analogues of triostin A.

Two new analogues of TANDEM (des-N-tetramethyl triostin A) have been synthesised in an effort to elucidate the molecular basis of DNA nucleotide sequence recognition in this series of compounds. Their binding preferences have been investigated by DNAase I footprinting and differential inhibition of restriction nuclease attack. The presence of a single N-methyl group on only one valine residue (in [N-MeVal4] TANDEM) abolishes the ability to recognise DNA, presumably because this antibiotic analogue has suffered an unfavourable conformational change in the depsipeptide ring. A bis-methylated analogue, [N-MeCys3, N-MeCys7]TANDEM, was found to interact quite strongly with DNA and afforded binding sites, rich in AT residues, identical to those of TANDEM. Footprinting with various DNA fragments of known sequence showed that this analogue recognises sequences containing the dinucleotide TpA, although we cannot exclude the possibility that it binds to ApT as well. [N-MeCys3, N-MeCys7]TANDEM inhibits cutting by RsaI, a restriction enzyme that recognises GTAC but not by Sau3AI which recognises GATC. This provides further supportive evidence that the ligand (and, by extension, TANDEM itself) prefers binding to sequences containing the dinucleotide step TpA.     

Cloning, sequencing and expression of a sialidase gene from Clostridium sordellii G12

A 4.3 kb XbaI restriction fragment of DNA from Clostridium sordellii G12 hybridized with a synthetic oligonucleotide representing the N-terminus of the sialidase protein secreted by C. sordellii. This cloned fragment was shown to encode only part of the sialidase protein. The sialidase gene of C. sordellii was completed by a 0.7 kb RsaI restriction fragment overlapping one end of the XbaI fragment. After combining the two fragments and transformation of Escherichia coli, a clone that expressed sialidase was obtained. The nucleotide sequence of the sialidase gene of C. sordellii G12 was determined. The sequence of the 18 N-terminal amino acids of the purified extracellular enzyme perfectly matched the predicted amino acid sequence near the beginning of the structural gene. The amino acid sequence derived from the complete gene corresponds to a protein with a molecular mass of 44,735 Da. Upstream from the putative ATG initiation codon, ribosomal-binding site and promoter-like consensus sequences were found. The encoded protein has a leader sequence of 27 amino acids. The enzyme expressed in E. coli has similar properties to the enzyme isolated from C. sordellii, except for small differences in size and isoelectric point. Significant homology (70%) was found with a sialidase gene from C. perfringens.     

Transforming growth factor-alpha: characterization of the BamHI, RsaI, and TaqI polymorphic regions.

We have characterized the nature of structural alleles of the transforming growth factor-alpha (TGF alpha) locus by restriction-enzyme digestion with BamHI, RsaI, and TaqI. The BamHI polymorphic site is located within exon VI, which codes for the 3' untranslated region. The two BamHI alleles differ by a single point mutation at the restriction site. The RsaI and TaqI polymorphic sites are located within intron V. The two alleles differ at the restriction site, either by a point mutation (RsaI) or by a 4-bp deletion (TaqI). This analysis permits us to devise a PCR method coupled with restriction digestions to directly identify the TGF alpha polymorphisms. Analysis of 99 Caucasian controls has revealed a highly significant (P < .001) association between the RsaI and the BamHI genotype. The frequency of the rare BamHI allele was significantly higher (P < .001) in transformed cell lines (.30) than in controls (.076).     

Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI.     

A new approach in recognition of heterochromatic regions of human chromosomes by means of restriction endonucleases.

The heteromorphisms of C-band regions of human chromosomes are evaluated by means of restriction endonucleases AluI, DdeI, MboI, and RsaI. Every chromosome exhibits heteromorphic markers of the C-band regions except chromosome 8. Each enzyme was found to be highly characteristic in its staining profile, a result that clearly suggests the diversity of heterochromatin. The inherent C-band-region heterochromatin variability that is revealed by these enzymes provides a valuable tool in identifying markers as compared with other previously described techniques.     

Association of DNA polymorphisms of the growth hormone and prolactin genes with milk productivity in Yaroslavl and black-and-white cattle

Polymorphisms of the prolactin (bPRL) and growth hormone (bGH) genes were studied comparatively in the Russian and German Black-and-White and Yaroslavl cattle breeds. Two polymorphisms were studied for each gene. In the case of the bPRL gene, the polymorphism of the 5'-untranslated region was examined by microsatellite analysis and the RsaI polymorphism of exon 3, by RFLP analysis. In the case of the bGH gene, the MspI polymorphism of intron III and the AluI polymorphism of exon 5 were assessed by RFLP analysis. Differences in allele and genotype frequencies were observed both between and within breeds. The heterozygosity at the RsaI marker was low (9.4%) in the Russian Black-and-White breed; that at the microsatellite of the bPRL gene was low (3.2-24%) in all breeds examined. Homozygotes BB at the bPRL gene, which had not been reported earlier for European cattle breeds, were detected in the German Black-and-White and Yaroslavl breeds (at frequencies 0.16 and 0.13, respectively). The frequency of allele MspI(-) of the bGH gene in the Yaroslavl breed was extremely low (0.02), comparable only with that of the Holstein cattle (0.02). The heterozygosity at the AluI polymorphism was higher than at the MspI polymorphism of the bGH gene and reached 55% in the Yaroslavl breed. Genotype BB of the RsaI polymorphism of the bPRL gene tended to show a negative association with the fat content in milk. The genotypes of the AluI polymorphism of the bGH gene were associated with the fat content in milk in the Yaroslavl (F = 4.56, P = 0.013) and German Black-and-White (F = 4.1, P = 0.014) breeds: the highest fat content in milk was observed in the subsample of cows with heterozygous genotype VL.     

Relationship between MTNR1A melatonin receptor gene polymorphism and seasonal reproduction in different goat breeds

The reproductive activity of goats bred in temperate latitude follows a seasonal pattern, influenced by annual variation in day length. Daily variation in pineal melatonin secretion is the neuroendocrine signal recognized by animals through the link between this hormone and melatonin receptor 1a (MTNR1A). A total of 345 goats of different breeds (225 Sarda, 30 Saanen, 30 Chamois Coloured, 30 Maltese and 30 Nubian) with a kidding period in October-December or January-March were analysed to verify if a link exists between the structure of the receptor gene and reproductive activity. The main part of exon II of MTNR1A gene was amplified by PCR and then digested with MnlI and RsaI to prove the presence of restriction sites. Sequencing of 20 cloned samples and 20 purified samples permitted comparison with previously published sequences. No polymorphism was found using MnlI enzyme, as all 345 samples showed the cleavage site in position 605 and all the goats were MM genotype. However, using RsaI enzyme, some Sarda goats, showed a polymorphic site in position 53. Nine Sarda goats were R/r genotype, lacking this cleavage site only in one allele, while the other animals, both Sarda and the other breeds, presented the cleavage site in both the alleles and were thus R/R genotype. No r/r genotype was found in any of the breeds. In Sarda goats the allelic frequency was 0.98 for R allele and 0.02 for r allele; genotypic frequency was 96.00% for R/R genotype and 4.00% for R/r genotype. A strong link emerged from statistical analysis (P<0.001) between R/r genotype and reproductive activity, which was strongly influenced by photoperiod. Sequencing indicated six nucleotide changes that did not induce any amino acid change. Data showed that polymorphism was present and that it influences reproductive activity only in the Sarda breed.     

Close linkage between bovine prolactin and BoLA-DRB3 genes: genetic mapping in cattle by single sperm typing.

The major histocompatibility complex and prolactin (PRL) genes are syntenic in humans and cattle but the genetic distance between these loci has not been determined for either species. In this study, the sperm typing technique was used to measure the recombination frequency between the bovine lymphocyte antigen (BoLA)-DRB3 and PRL loci. A total of 300 sperm were typed from one doubly heterozygous bull for segregation of DRB3 and PRL alleles. Sperm typing was performed using the polymerase chain reaction (PCR) and restriction enzyme cleavage of the PCR products, followed by resolution of the restriction fragments in polyacrylamide gels. Digestion with the restriction endonuclease RsaI allowed the unambiguous discrimination of alleles for both loci. The maximum likelihood estimation of the recombination fraction theta = 0.04, with a 95% confidence interval of 0.01 to 0.07. Close linkage between PRL and DRB3 has important implications for marker-assisted selection in animal breeding since PRL has been shown to be closely linked to a locus that affects milk yield, and BoLA loci influence susceptibility to a number of infectious diseases. Our results demonstrate the general applicability of the sperm typing procedure for gene mapping in species other than humans and provide an example of how parallel efforts to map the genomes of agriculturally important species of animals can have a positive impact on the development of a primary human linkage map.     

Site of the base change in the vaccinia virus DNA polymerase gene which confers aphidicolin resistance.

An aphidicolin-resistant mutant of vaccinia virus has been shown to encode an altered viral DNA polymerase that is more resistant to aphidicolin. Marker transfer experiments with the DNA from the resistant virus localized the mutation site to an RsaI segment within the portion of the HindIII-E segment which has been shown to contain the viral DNA polymerase gene. Nucleotide sequence analysis of the mutant DNA showed a single GC to AT transition at position 2430, which indicates a leucine-to-methionine change at residue 645 in the protein.     

DNA polymorphism in the Mongolian population. Restriction fragment length polymorphism analysis of mitochondrial DNA]

Restriction enzyme fragment patterns in the D loop and deletion-insertion polymorphism in the V noncoding region of human mitochondrial DNA (mt DNA) were analysed in Mongolian population using the polymerase chain reaction. Polymorphisms were detected and mt DNAs classified into 40 types using seven enzymes--AvaII, BamHI, CfrI131, KpnI, EcoRV, HaeIII RsaI and Asian specific deletion and insertion. The allele frequencies of the polymorphisms and gene diversity were determined. The data obtained for Mongolian population and the literature data were comparatively studied.     

Correlation between RsaI restriction fragment length polymorphism and electrophoretic types of human placental alkaline phosphatase

Restriction fragment length polymorphism (RFLP) of human alkaline phosphatases was studied in a population sample from northern Sweden using a placental alkaline phosphatase (PLAP) cDNA probe. After digestion of human genomic DNA with RsaI the Southern blots showed DNA fragments most probably derived from three genes: PLAP, germ cell alkaline phosphatase (PLAP-like) and intestinal alkaline phosphatase. In agreement with a previous study, a two-allele polymorphism was found in PLAP with bands at 1.6 kilobases (A1) and 1.8 kilobases (A2). The gene frequencies of A1 and A2 were 0.46 and 0.54, respectively. There was a significant correlation between the RsaI RFLPs and electrophoretic types of PLAP; RSAI A2 showed an association with the ALP2p allele of PLAP.