Restriction Enzyme Site Polymorphisms Research Articles

Taxonomic Studies of Rickettsiella, Rickettsia, and Chlamydia Using Genomic DNA

Invertebrate pathogens of the genus Rickettsiella (Order Rickettsiales) undergo a developmental cycle more characteristic of chlamydial organisms (Order Chlamydiales) than of typical rickettsiae. Moreover, among recognized species of Rickettsiella, there is considerable variation in host range and in the ultrastructure and development of the infectious stage, i.e., the elementary body. To begin an analysis of the taxonomic relationships of the invertebrate pathogens belonging to the genus Rickettsiella and of the relationship of these to chlamydiae and other rickettsiae, genomic DNA of representative species was compared with respect to restriction enzyme site polymorphism, DNA-DNA hybridization, G + C ratios, and fine melting profiles. The strains studied included isolates currently classified as Rickettsiella grylli, R. popilliae, R. chironomi, Chlamydia psittaci, C. trachomatis, Coxiella burnertii, and Rickettsia conorii. Reciprocal DNA hybridization studies carried out under high-stringency conditions (65°C) showed homology between R. grylli and R. popilliae isolates but not with the others, including two isolates of R. chironomi from the midge, Chironomus dorsalis, and the scorpion, Buthus occitanus. The R. chironomi isolates shared no detectable homology with each other or with any of the other strains or species. Additionally, no homology was detected between any of the Rickettsiella isolates and the species of Coxiella, Rickettsia, or Chlamydia. Results obtained from the DNA fine melting profiles and G + C ratios corresponded with the results obtained from the DNA hybridization studies. Thus, the present study indicates that, despite similarities in developmental cycles, Rickettsiella and Chlamydia are taxonomically distinct groups appropriately placed in different orders. However, the uniqueness of the two R. chironomi species, both from each other and from the other organisms studied, indicates these could be recognized as distinct species.

A polymerase chain reaction‐based method for the detection of trisomy 12 in B chronic lymphocytic leukaemia

We report a polymerase chain reaction (PCR)-based method for detection of clonal trisomy 12 cell populations in B cell chronic lymphocytic leukaemia which is particularly applicable to retrospective analysis or archival material. The method utilizes three restriction enzyme site polymorphisms linked to the KRAS2 locus on chromosome 12 to monitor allele imbalance resulting from duplication of this chromosome. 21/24 unselected chronic lymphocytic leukaemia cases (88%) were informative for at least one polymorphism and of these, eight (33% of total cases) displayed allelic imbalance consistent with trisomy 12.

Beta-globin gene cluster haplotypes in Yanomama Indians from the Amazon region of Brazil.

Six polymorphic restriction enzyme sites in the beta-globin gene cluster were investigated in Yanomama Indians from the Amazon region of Brazil, using the polymerase chain reaction (PCR) technique. Four haplotypes were identified; the haplotype frequency distribution is similar to those reported for Polynesians, Micronesians and most Asian populations.

Restriction enzyme analysis of the chloroplast and nuclear 45s ribosomal DNA ofAllium sectionsCepa andPhyllodolon (Alliaceae)

Estimates of the phylogenetic relationships among cultivated and wildAllium species would benefit from identification of molecular characters. Restriction enzyme analysis of the chloroplast DNA (cpDNA) of the bulb onion (Allium cepa), Japanese bunching onion (A. fistulosum), wildAllium species in sect.Cepa andPhyllodolon, and the outgroupsA. ampeloprasum andA. tuberosum detected 39 polymorphisms.Allium cepa andA. vavilovii were identical for all characters. Cladistic analysis generated three most-parsimoniousWagner trees of 44 steps differing only in a zero-length branch.Allium fistulosum andA. altaicum (sect.Phyllodolon) comprised a monophyletic lineage separated from theA. cepa andA. vavilovii of sect.Cepa. The unresolved node was composed ofA. galanthum, A. roylei, and the lineage containingA. cepa, A. vavilovii, A. fistulosum, andA. altaicum. The clade containingA. altaicum, A. cepa, A. fistulosum, A. galanthum, A. roylei, andA. vavilovii remained resolved for strict consensus ofWagner trees of 48 steps or less.Allium pskemense andA. oschaninii were increasingly distant.Allium oschaninii has been proposed as the progenitor of the bulb onion, but was more closely related to the common progenitor of all species in sect.Cepa andPhyllodolon. Phylogenies estimated from cpDNA characters usingDollo parsimony resulted in a single most-parsimonious tree of 46 steps and agreed with phylogenies based onWagner parsimony. Polymorphic restriction enzyme sites in the 45s ribosomal DNA were not used to estimate phylogenies because of uncertain homologies, but are useful for identifying interspecific hybrids. The maternal phylogenies estimated in this study help to distinguish wildAllium species closely related to the bulb onion. Although not in agreement with classifications based on morphology, the phylogenies closely reflected crossability among species in sect.Cepa andPhyllodolon.

Restriction enzyme analysis of the nuclear 45s ribosomal DNA of six cultivated Alliums (Alliaceae)

Estimates of the phylogenetic relationships among cultivated and wildAllium species would benefit from identification of objective molecular characters. Restriction fragment length polymorphisms in the nuclear 45s ribosomal DNA (rDNA) were identified among two of five accessions of each of six cultivated Alliums. Restriction enzyme sites forBamHI,DraI,EcoRI,EcoRV,SacI, andXbaI were mapped. Different lengths of the rDNA repeat unit among the cultivated Alliums were due to sizes of the intergenic spacer. Nineteen polymorphic restriction enzyme sites were discovered and used to estimate phylogenetic relationships. Cladistic analyses based on Wagner parsimony were completed without an outgroup and resulted in two equally most parsimonious trees of 22 steps. A combined analysis of differences at RE sites in the ribosomal (19 characters) and chloroplast (15 characters) DNA generated a single most parsimonious tree of 39 steps. Single trichotomies were observed at 40 and 41 steps. Strict consensus of the three trees of 41 or fewer steps consisted of a lineage forA. tuberosum, a second forA. ampeloprasum andA. sativum, and a third forA. cepa, A. fistulosum, andA. schoenoprasum. Estimates of phylogenetic relationships based on variability at restriction enzyme sites in the rDNA and chloroplast DNA agree with the classification scheme ofTraub. Because of the predominance of autapomorphies, restriction enzyme analysis of the nuclear 45s rDNA is of limited use in estimating phylogenies amongAllium sections. However it is useful in the establishment of interspecific hybridity.

Application of PCR amplification of DNA from paraffin embedded tissue sections to linkage analysis in familial retinoblastoma.

A family segregating for the retinoblastoma predisposition gene has been analysed using the polymerase chain reaction to exclude their son as being an affected gene carrier. The unusual feature of this family is that the affected child, who would ordinarily have been used to establish phase in a linkage study, died as a result of developing a second tumour some years ago. The only tissue available from this child was a paraffin embedded, formalin fixed histopathological specimen from the second tumour. It was possible to isolate DNA from this tissue and amplify the DNA flanking two polymorphic restriction enzyme sites to establish alleles which cosegregated with tumour predisposition. Archival material can now be used to offer families such as this prenatal screening to provide informed genetic counselling.

DNA Sequence, genomic organization, and chromosomal localization of the mouse peripheral myelin protein zero gene: Identification of polymorphic alleles

We have cloned and characterized the mouse gene, P 0, that encodes the predominant protein of peripheral myelin. Similar to the rat gene, the mouse P 0 gene is encoded by six exons that span about 7 kb of DNA. The DNA sequence of the mouse gene is highly homologous with the rat gene, including the regions believed to be important in transcriptional control. Furthermore, the P 0 protein appears well conserved throughout evolution. The gene was mapped to mouse chromosome 1 by Southern analysis of a Chinese hamster × mouse somatic cell hybrid panel. Several polymorphic restriction enzyme sites were identified within the P 0 locus. Recombinant inbred strain mapping has linked the P 0 gene to Ly-9/Ly-17/Sap in a region corresponding to band 1H3.

A rapid method for genotyping mice for T cell receptor V βa and V βb haplotypes by PCR analysis of whole blood

Strains of laboratory mice bearing a germline deletion of some T cell receptor V β genes have proven useful in a variety of studies of T cell receptor function. Analysis of genetic crosses between deleted and wild type strains can provide information about the relevance of genes located within the deletion to specific T cell responses. Existing techniques for genotyping offspring of such crosses usually involve flow cytometric analysis which may not be available to all laboratories. Recent nucleotide sequence data indicate the presence of two restriction enzyme site polymorphisms in the closely linked V β1 gene which discriminate between deleted and wild-type strains. Amplification of DNA segment containing the diagnostic sites by polymerase chain reaction followed by restriction enzyme digestion of the product offers a simple and rapid method for genotyping animals.

Cloning and characterization of the rRNA genes and flanking regions from Babesia bovis: use of the genes as strain discriminating probes

Three sets of rRNA genes (units 1S–3S) have been identified in Babesia bovis (Samford isolate). All three units are present in the same, probably single, copy number. The rRNA genes and flanking regions have been analysed by cloning, restriction mapping and DNA hybridization. The units are approximately 7 kb in length and have essentially identical restriction maps. In contrast the flanking regions exhibit significant restriction site differences. However, the regions upstream of all three units are related and sequences similar to part of the region upstream of units 1S and 3S are present in multiple copies in the genome. The downstream regions appear to be unrelated, but downstream from unit 1S is a region of at least 7 kb similar to a second region not closely linked to the rDNA units. The restriction enzyme site polymorphisms in the flanking regions of the equivalent units in different isolates allow ready discrimination among six different isolates of B. bovis.

Mitochondrial DNA of Physarum polycephalum: Physical mapping, cloning and transcription mapping

Mitochondrial DNA (mtDNA) has been isolated from four strains of Physarum polycephalum and a restriction site map has been determined using nine restriction enzymes. The restriction site maps of the four strains are similar but each strain is distinguished by insertions, deletions and restriction enzyme site polymorphisms. The sum of the restriction fragments gives mitochondrial genome sizes which vary from about 56 kb to 62 kb. In all four strains the composite map of the restriction enzyme sites for the mtDNA is circular. Knowledge of the restriction enzyme map has enabled cloning of mtDNA fragments representing the entire mtDNA of strain M3. The cloned fragments have been used to create a transcription map of the mtDNA.

Kappa Light Chain Gene Rearrangement in T-Cell Acute Lymphoblastic Leukemia

Immunoglobulin and T-cell receptor gene rearrangement assays are sensitive methods of detecting clonality in lymphoproliferative disorders. The lack of lineage specificity of immunoglobulin heavy chain and T-cell receptor beta and gamma gene probes in acute leukemia is well established. However, immunoglobulin light chain gene rearrangement traditionally has been considered a highly specific indicator of a clonal B-lineage process. The authors describe a case of T-cell acute lymphoblastic leukemia in which Southern blot hybridization studies showed rearrangement of the T-cell receptor beta chain gene. Unexpectedly, the immunoglobulin kappa light chain gene also was rearranged; no immunoglobulin heavy chain gene rearrangement was seen. Kappa rearrangement was confirmed with the use of three restriction endonucleases. No rearrangements were seen from normal skin tissue, making a restriction enzyme site polymorphism highly unlikely. Northern blot studies showed a normal-size, T-cell receptor beta chain gene transcript; no immunoglobulin RNA was identified. These results describe the first reported case of kappa light chain gene rearrangement in a T-cell acute lymphoblastic leukemia. The findings emphasize the necessity of interpreting molecular hybridization studies in conjunction with routine morphology and immunophenotyping studies.

The abnormal oocyte phenotype is correlated with the presence of blood transposon in Drosophila melanogaster.

The abnormal oocyte mutation (2;44) originates in the wild: it confers no visible phenotype on homozygous abo males or females, but homozygous abo females produce defective eggs and the probability of their developing into adults is much lower than that of heterozygous sister females. We isolated by chromosome walking 200 kb of DNA from region 32. This paper reports that a restriction enzyme site polymorphism analysis in wild type and mutant stocks allowed us to identify a DNA rearrangement present only in stocks carrying the abo mutation. The rearrangement is caused by a DNA insert on the abo chromosome in region 32E which, by restriction map and sequence analysis, was identified as copia-like blood transposon. The transposon, in strains that had remained in abo homozygous conditions for several generations and had lost the abo maternal-effect, was no longer present in region 32E. Certain features of the abo mutation, discussed in the light of this finding, may be ascribed to the nature of the particular allele studied.

Haemophilia A: carrier detection by DNA analysis.

From 46 families of predominantly German origin, afflicted with haemophilia A, 178 females were tested for carrier status. Two polymorphic restriction endonuclease sites, the extragenic marker locus DXS 52 (St 14 probe) and the intragenic Bcl I RFLP were investigated in these families. In some cases the results were corroborated by identifying (i) deletions within the factor VIII:C gene and (ii) eliminating a restriction endonuclease site. Two new alleles of the DXS 52 marker locus were found. According to this strategy, 27 women were classified as carriers and 74 as non-carriers. Forty-six women were classified as carriers according to pedigree analysis. Twenty-five females of families with sporadic cases and 6 test persons, who had mothers who where homozygous for the marker alleles, were diagnosed by additional use of conventional carrier detection.

Non-deletion haemoglobin H disease in Papua New Guinea.

Analysis of DNA from members of a Melanesian family from Papua New Guinea with haemoglobin (Hb) H disease revealed that all four alpha globin genes are intact in affected subjects. Study of restriction enzyme site and length polymorphisms and the use of oligonucleotide probes indicated that the molecular basis of this Papuan form of non-deletion Hb H disease differs from the previously described Middle Eastern and Mediterranean types.

Polymorphic DNA haplotypes at the human phenylalanine hydroxylase locus and their relationship with phenylketonuria.

Eight polymorphic restriction enzyme sites at the phenylalanine hydroxylase (PAH) locus were analyzed from the parental chromosomes in 33 Danish nuclear families with at least one phenylketonuric (PKU) child. Determination of haplotypes of 66 normal chromosomes and 66 chromosomes bearing mutant allele(s) demonstrated that there are at least two haplotypes which occur predominantly on PKU chromosomes and rarely otherwise. Overall, the relative frequencies of the various haplotypes are significantly different on PKU- and normal-allele bearing chromosomes, even though there is no predominantly occurring unique haplotype which can characterize the PKU chromosomes. In addition, no significant association (linkage disequilibrium) between any single polymorphic site and the mutant allele(s) was observed. The results suggest that either the phenylketonuric mutation was very ancient so that the polymorphic sites and the mutation have reached linkage equilibrium or the mutant allele(s) are the results of multiple mutations in the phenylalanine hydroxylase gene in man. Furthermore, a crude relationship between standardized linkage disequilibria and physical map distances of the polymorphic sites indicates that there is no apparent recombination hot-spot in the human phenylalanine hydroxylase gene, since the recombination rate within the locus appears to be uniform and likely to be occurring at a rate similar to that within the HLA gene cluster. The limitations of this later analysis are discussed in view of the sampling errors of disequilibrium measure used, and the potential utility of the PAH haplotypes for prenatal diagnosis and detection of PKU carriers is established.

A benign form of thalassaemia intermedia may be determined by the interaction of triplicated α locus and heterozygous β-thalassaemia

In this paper we report that the combination of a triplicated alpha globin locus with heterozygous beta-thalassaemia produces a clinical phenotype of thalassaemia intermedia in five Italian subjects from four unrelated families, while in two other cases the phenotype was thalassaemia minor. The haematological findings of the five patients were uniform, producing a benign form of thalassaemia intermedia, transfusion independent, with a long life expectancy. The pattern of inheritance of the two genetic determinants and the more pronounced beta/alpha globin chain imbalance, demonstrates that the genetic combination is indeed the cause of the phenotype. The pattern of restriction enzyme site polymorphisms suggests the presence of the beta IVS I 110 G----A mutation at least in three of these cases.

Structural Analysis of the Polymorphic 3′ Region of the Human Chorionic Gonadotropin-αGene in Normal Placenta and Choriocarcinoma Cells*

We previously examined the segregation patterns of two polymorphic restriction enzyme sites for EcoRI and HindIII in the 3'-flanking region of human CG-alpha gene in placenta and choriocarcinoma. We observed that a unique combination of restriction sites predominates in choriocarcinoma while this pattern was rare in placenta, suggesting a functionally important site in this region of genome. Genomic libraries were constructed from DNA derived from placenta and from the choriocarcinoma cell line JAr to examine the DNA sequence responsible for these polymorphic sites. Several clones were isolated; restriction fragments containing the polymorphic sites were subcloned into pBR322, and the stretch of DNA encompassing the polymorphic sites were sequenced. No apparent DNA rearrangements were observed; sequence analysis showed that both of the polymorphic sites were caused by single base pair mutation in the recognition sequence of each enzyme. Three new polymorphic sites were identified in the 3' region of the hCG-alpha gene. These data indicate that major DNA rearrangements in the 3' region of the gene are not associated with choriocarcinoma. However, analysis of the polymorphic sites revealed that there is a linkage disequilibrium among these sites which may extend to a putative oncogene which is responsible for developing choriocarcinoma.

Molecular analysis of the hotspot of recombination in the murine major histocompatibility complex.

Biological and serological assays have been used to define four subregions for the I region of the major histocompatibility complex (MHC) in the order I-A, I-B, I-J, and I-E. The I-J subregion presumably encodes the I-J polypeptide of the elusive T-cell suppressor factors. Restriction enzyme site polymorphisms and DNA sequence analyses of the I region from four recombinant mouse strains were used to localize the putative I-B and I-J subregions to a 1.0-kilobase (kb) region within the E beta gene. Sequencing this region from E beta clones derived from the two mouse strains: B10.A(3R), I-Jb and B10.A(5R), I-Jk initially used to define the I-J subregion revealed that these regions are identical, hence the distinct I-Jb and I-Jk molecules cannot be encoded by this DNA. In addition, the DNA sequence data also refute the earlier mapping of the I-B subregion. Analysis of the DNA sequences of three parental and four I region recombinants reveals that the recombinant events in three of the recombinant strains occurred within a 1-kb region of DNA, supporting the proposition that a hotspot for recombination exists in the I region. The only striking feature of this hotspot is a tetramer repeat (AGGC)n that shows 80 percent homology to the minisatellite sequence which may facilitate recombination in human chromosomes.

The evolution of the ?- and ?-globin gene clusters in human populations

DNA analysis of the alpha- and beta-globin gene clusters has revealed substantial variability between individuals and populations. As well as restriction enzyme site and length polymorphisms, variation in gene copy number and type is observed. Because of this extensive polymorphism DNA analysis offers a highly informative method of studying genetic affinities between human populations. Haplotypes, consisting of a set of restriction enzyme polymorphisms distributed along the cluster, have been developed for both loci. Analysis of the molecular basis of numerous beta-thalassaemia alleles has revealed, in general, different sets of mutations in different populations, indicating that these postdate the racial divergence. Recent microepidemiological studies on the distribution of alpha-thalassaemia support the hypothesis that this condition, like the beta s-mutation, has been selected because it confers protection against malaria. Population-specific DNA polymorphisms at these and other loci promise to be of considerable value to genetic anthropology.

Restriction fragment-length polymorphisms of class II gene sequences in mice expressing minor structural variants of I-Ak and I-Ap.

Serologic and structural analyses of the I-A molecules expressed among a large collection of wild mouse-derived H-2 haplotypes has led to the definition of "families" of I-A alleles which encode antigenically similar molecules that are identical in more than 90% of their tryptic peptides. Two of these families, denoted the I-Ak and I-Ap families, consist of 10 I-A alleles which encode I-A molecules whose structures are closely related to either I-Ap or I-Ak. The evolutionary relationships of the I-A alleles in these families were assessed by a molecular analysis of their genomic structures. The A alpha and A beta alleles within these I-A families were compared by analysis of restriction fragment-length polymorphisms (RFLP) detected at high stringency by Southern blot hybridization with DNA probes specific for either A alpha or A beta. The polymorphic restriction enzyme sites detected in this survey were distributed over more than 7 kb of genomic DNA surrounding each gene. Because both A alpha and A beta are encoded by about 700 bp of exon DNA, the majority of the restriction enzyme sites assayed by this RFLP analysis reflect polymorphisms in noncoding regions. The DNA sequence homologies of these alleles were estimated from the RFLP results with seven restriction endonucleases by calculating the fraction homologous value as defined previously. The results indicate that evolutionarily dissimilar I-A alleles can encode I-A molecules with very similar structures. The five I-A alleles in the I-Ak family could be divided into two discrete groups, denoted K1 and K2, on the basis of their restriction fragment (RF) genotypes. The RF genotypes of alleles within each group shared more than 80% of the restriction fragments for both A and A beta. In contrast, the RF genotypes of alleles in group K1 differed extensively from those in group K2, indicating that alleles in these separate groups may not be evolutionarily closely related. These observations suggest that gene conversion or intragenic recombinational events may have been involved in the evolution of groups K1 and K2 in the I-Ak family. The RF genotypes of alleles in the I-Ap family demonstrated a close evolutionary relationship among all but two of the alleles. These two alleles encoded I-A molecules whose structures were the least related to I-Ap of any of the alleles in the I-Ap family.(ABSTRACT TRUNCATED AT 400 WORDS)