Variability for restriction fragment lengths and phylogenies in lentil

Summary Thirty accessions of domesticated (Lens culinaris ssp. culinaris) and wild (L. culinaris ssp. orientalis, L. culinaris ssp. odemensis, L. nigricans ssp. ervoides and L. nigricans ssp. nigricans) lentil were evaluated for restriction fragment length polymorphisms (RFLPs) using ten relative low-copy-number probes selected from partial genomic and cDNA libraries of lentil. Nei's average gene diversity was used as a measure of genetic variability for restriction fragment lengths within subspecies and a dendrogram was constructed from genetic distance estimates between subspecies. The wild lentils L. culinaris ssp. orientalis and L. culinaris ssp. odemensis showed the greatest variability for restriction fragment lengths and were closely positioned to domesticated lentil in the dendrogram. Little variability for restriction fragment lengths was observed within accessions of L. nigricans ssp. ervoides and L. nigricans ssp. nigricans. This observation is consistent with a previously published proposal that nigricans may have been independently domesticated. Estimates of genetic variability based on RFLPs tended to be greater than estimates from isozymes.     

RFLP of 16S-ITSrDNA Region to Differentiate Lactobacilli at Species Level

The 16S-ITS (internal transcribed spacer) region of the rrn operon was amplified by polymerase chain reaction (PCR). The amplification products were analysed by restriction fragment length polymorphism (RFLP) using a set of restriction enzymes, AluI, HaeIII, and TaqI. Restriction pattern analyses revealed that TaqI restriction enzyme could clearly differentiate the nine reference strains of Lactobacillus used in the study. This revised version was published online in July 2006 with corrections to the Cover Date.     

SstII polymorphic sites in the promoter region of the human cystatin C gene

Summary By direct sequencing of polymerase chain reaction (PCR) amplified DNA from different individuals, three point mutations have been found in a 220-bp fragment from the promoter region of the human cystatin C gene. The three mutations are all localized within a short segment of 85 bp on the same allele. One of the base substitutions results in the generation of a novel SstII restriction site and another in the loss of the commonly occurring SstII restriction site. A PCR-based assay for analysis of the two SstII sites was designed and used to demonstrate Mendelian inheritance of the polymorphism. This SstII restriction fragment lenght polymorphism offers a new probe-independent marker for chromosome 20 linkage studies.     

Identification of a second polypeptide required for McrB restriction of 5-methylcytosine-containing DNA in Escherichia coli K12

Summary The McrB restriction system in Escherichia coli K12 causes sequence-specific recognition and inactivation of DNA containing 5-methylcytosine residues. We have previously located the mcrB gene near hsdS at 99 min on the E. coli chromosome and demonstrated that is encodes a 51 kDa polypeptide required for restriction of M.AluI methylated (A-G-5mC-T) DNA. We show here, by analysis of maxicell protein synthesis of various cloned fragments from the mcrB region, that a second protein of approximately 39 kDa is also required for McrB-directed restriction. The new gene, designated mcrC, is adjacent to mcrB and located distally to hsdS. The McrB phenotype has been correlated previously with restriction of 5-hydroxy-methylcytosine (HMC)-containing T-even phage DNA that lacks the normal glucose modification of HMC, formally designated RglB (for restriction of glucoseless phage). This report reveals a difference between the previously correlated McrB and RglB restriction systems: while both require the mcrB gene product only the McrB system requires the newly identified mcrC-encoded 39-kDa polypeptide.     

The plastome mutator of Oenothera continues to function as originally described

Summary Recently, Lindenhahn et al. (1985) hypothesized that the plastome mutator (pm) system in Oenothera originated through contaiminating cross-pollination and that the variegation was an example of hybrid plastome-genome incompatibility. Their evidence was based on restriction pattern analyses of white sectors which showed wild-type plastome III patterns rather than the wild-type plastome I patterns of the green portions of their plants. Their hypothesis does not adequately account for the results which our laboratories have obtained independently; the pm-system of Oenothera continues to generate many new and different plastome mutations following the genetic parameters as published originally (Epp 1973). Our studies support mutator gene function. The restriction pattern of the chloroplast DNA of five newly isolated pm-induced variegation sectors are reported here to show a restriction pattern identical to the green wild-type plastids. The restriction pattern reported by Lindenhahn et al. (1985) for their white sector plastids is different than we would expect from a pm-induced plastome mutation. Their overall analysis did not utilize many of the salient features of the genetics of Oenothera and of the pm-system. The white sectors they observed are probably due to an accidental contamination by plastome III plastids. Suggestions are made for delineating experimentally plastome mutations and hybrid incompatibility. For future analyses, a comparative study of numerous pm-induced sectors is recommended, since the pm-system readily generates many different plastome mutations with independent origins. This comparison would greatly assist in the interpretation of restriction patterns.     

Molecular phylogeny of mangroves VII. PCR-RFLP of trnS-psbC and rbcL gene regions in 24 mangrove and mangrove-associate species

Chloroplast DNA (cpDNA) regions, trnS-psbC and rbcL, from 120 individuals of 24 mangrove and mangrove associate species belonging to 11 orders, 13 families and 17 genera of Angiospermae were amplified by the polymerase chain reaction (PCR) and restriction-digested with HaeIII. Analysis of polymorphism in the restriction fragments (PCR-RFLP) revealed 18 classes of restriction banding pattern in trnS-psbC region. This has provided molecular evidence for diversity in the mangrove floral component at the above-species level. Intra-generic variations were observed in three genera, viz. Rhizophora, Avicennia and Suaeda. Species-specific restriction patterns were found in the genera Rhizophora and Suaeda. A natural hybrid belonging to the genus Rhizophora was also analysed, and its restriction pattern was the same as that of a putative parental species.PCR-RFLP analysis of rbcL gene region was less differentiating. However, it showed 13 different classes of restriction patterns and revealed the usefulness of these investigations for genome analysis at a higher taxonomic level. Intra-specific variation was not observed in any of the species in either of the cpDNA regions analysed. This is the first report which describes variations in the chloroplast genome of mangrove species. Received: 20 April 1999 / Accepted: 12 May 1999     

Heterozygosity and localisation of normal allelic fragments for an alpha1-antitrypsin homologous sequence

Summary Approximately 10 kb downstream of the alpha₁-antitrypsin (AAT) gene is a homologous sequence. Two polymorphisms detected with the restriction enzymes Bg1II and EcoRI have been reported previously. We describe two additional polymorphisms with the restriction endonucleases TaqI and HindIII and, for all four restriction enzymes, we have mapped the fragments corresponding to the normal alleles in a cosmid clone.     

Altered immune proteome of Staphylococcus aureus under iron‐restricted growth conditions

Staphylococcus aureus is one of the major causative agents of severe infections, and is responsible for a high burden of morbidity and mortality. Strains of increased virulence have emerged (e.g. USA300) that can infect healthy individuals in the community and are difficult to treat. To add to the knowledge about the pathophysiology of S. aureus, the adaption to iron restriction, an important in vivo stressor, was studied and the corresponding immune response of the human host characterized. Using a combination of 1D and 2D immune proteomics, the human antibody response to the exoproteomes of S. aureus USA300Δspa grown under iron restriction or with excess iron was compared. Human antibody binding to the altered exoproteome under iron restriction showed a 2.7‐ to 6.2‐fold increase in overall signal intensity, and new antibody specificities appeared. Quantification of the secreted bacterial proteins by gel‐free proteomics showed the expected strong increase in level of proteins involved in iron acquisition during iron‐restricted growth compared to iron access. This was accompanied by decreased levels of superantigens and hemolysins. The latter was corroborated by functional peripheral blood mononuclear cell proliferation assays. The present data provide a comprehensive view of S. aureus exoproteome adaptation to iron restriction. Adults have high concentrations of serum antibodies specific for some of the newly induced proteins. We conclude that iron restriction is a common feature of the microenvironment, where S. aureus interacts with the immune system of its human host.     

Oenothera chloroplast DNA polymorphisms associated with plastome mutator activity

Summary Oenothera plants homozygous for a recessive allele at the plastome mutator (pm) locus show non-Mendelian mutation frequencies that are 1000-fold higher than spontaneous levels. Chloroplast DNA (cpDNA) was isolated from nine mutants and two green isolates of the plastome mutator line. cpDNA restriction patterns were compared to cpDNA from a representative of the progenitor Johansen strain, and cpDNAs from all eleven plastome mutator lines show changes of fragment mobility due to deletion events at five discrete regions of the plastome. Most of the mutants have cpDNA restriction patterns identical to that of one of the green isolates from the plastome mutator line, and therefore, most of the differences in fragment length are probably not responsible for the mutant phenotypes. In contrast to the plastome mutator line, cpDNA from several populations of a closely related wild-type Oenothera species have few restriction fragment length polymorphisms. This suggests that both mutation frequencies and site-specific cpDNA deletions are elevated in the plastome mutator line, and implicates a defect in the cpDNA repair or replication machinery.     

Sir-2.1 modulates 'calorie-restriction-mediated' prevention of neurodegeneration in Caenorhabditis elegans: implications for Parkinson's disease

The phenomenon of aging is known to modulate many disease conditions including neurodegenerative ailments like Parkinson’s disease (PD) which is characterized by selective loss of dopaminergic neurons. Recent studies have reported on such effects, as calorie restriction, in modulating aging in living systems. We reason that PD, being an age-associated neurodegenerative disease might be modulated by interventions like calorie restriction. In the present study we employed the transgenic Caenorhabditis elegans model (P_dat-1::GFP) expressing green fluorescence protein (GFP) specifically in eight dopaminergic (DA) neurons. Selective degeneration of dopaminergic neurons was induced by treatment of worms with 6-hydroxy dopamine (6-OHDA), a selective catecholaminergic neurotoxin, followed by studies on effect of calorie restriction on the neurodegeneration. Employing confocal microscopy of the dopaminergic neurons and HPLC analysis of dopamine levels in the nematodes, we found that calorie restriction has a preventive effect on dopaminergic neurodegeneration in the worm model. We further studied the role of sirtuin, sir-2.1, in modulating such an effect. Studies employing RNAi induced gene silencing of nematode sir-2.1, revealed that presence of Sir-2.1 is necessary for achieving the protective effect of calorie restriction on dopaminergic neurodegeneration.Our studies provide evidence that calorie restriction affords, an sir-2.1 mediated, protection against the dopaminergic neurodegeneration, that might have implications for neurodegenerative Parkinson’s disease.     

Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region

In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation. Received: 2 July 1997 / Accepted: 7 December 1997     

Chloroplast DNA evidence for introgression and long distance dispersal in the desert annualSenecio flavus (Asteraceae)

Phylogenetic analysis of chloroplast DNA (cpDNA) restriction site variation supports a close genetic relationship between the Southwest AsianSenecio flavus subsp.breviflorus and the North AmericanS. mohavensis. The intercontinental disjunct distribution of these two desert annuals may have originated via long distance dispersal. The chloroplast genomes of the Southern and North AfricanS. flavus subsp.flavus and subsp.breviflorus differ by at least ten restriction sites, while at most two restriction sites differentiate the cpDNA genomes of subsp.breviflorus and the outgroupS. squalidus. This suggests that the cpDNA genome ofS. flavus subsp.breviflorus may have resulted from introgression and chloroplast capture with a Mediterranean species related toS. squalidus. This hypothesized introgression could account for the morphological distinctiveness and duplicated isozyme loci ofS. flavus subsp.breviflorus relative to subsp.flavus.     

Abdominal Visceral Fat is Associated with a BclI Restriction Fragment Length Polymorphism at the Glucocorticoid Receptor Gene Locus

Several investigations have suggested that body fat distribution is influenced by nonpathologic variations in the responsiveness to Cortisol. Genetic variations in the glucocorticoid receptor (GRL) could therefore potentially have an impact on the level of abdominal fat. A restriction fragment length polymorphism (RFLP) has previously been detected with the BelI restriction enzyme in the GRL gene identifying two alleles with fragment lengths of 4.5 and 2.3 kb. This study investigates whether abdominal fat areas measured by computerized tomography (CT) are associated with this polymorphism in 152 middle-aged men and women. The less frequent 4.5-kb allele was found to be associated with a higher abdominal visceral fat (A VF) area independently of total body fat mass (4.5/4.5 vs. 2.3/2.3 kb genotype; men: 190.7 ± 30.1 vs. 150.7 ± 33.3 cm², p=0.04; women: 132.7 ± 37.3 vs. 101.3 ± 34.5 cm², p=0.06). However, the association with AVF was seen only in subjects of the lower tertile of the percent body fat level. In these subjects, the polymorphism was found to account for 41% (p=0.003) and 35% (p=0.007), in men and women, respectively, of the total variance in AVF area. The consistent association between the GRL polymorphism detected with BelI and AVF area suggests that this gene or a locus in linkage disequilibrium with the BelI restriction site may contribute to the accumulation of AVF.     

Phylogenetic relationships ofCrepidiastrum (Asteraceae) of the Bonin (Ogasawara) Islands based on chloroplast DNA restriction site variation

RFLPs of cpDNA were investigated for seven species ofCrepidiastrum, of which three are endemic to the Bonin Islands. As an outgroup for rooting the tree,Paraixeris denticulata was also examined. Approximately 350 restriction sites were surveyed using 16 restriction enzymes. A total of 26 restriction site mutations were detected, and seven of them were phylogenetically informative. Wagner parsimony analysis resulted in four most parsimonious trees. In the tree obtained, the Bonin endemics are monophyletic with four mutations and a bootstrap value of 0.98 for the branch. This result agrees with that obtained from a previous electrophoretic analysis (Ito and Ono 1990), and supports the hypothesis that the three Bonin endemics have been evolved from a common ancestor. The Bonin endemics cluster together withC. lanceolatum, suggesting that the endemics andC. lanceolatum share a common ancestor, although the bootstrap probability is not very high (0.78) and thus other possibilities cannot be rejected.     

Map location of the Escherichia coli origin of replication.

Summary Working with restriction fragments obtained directly from the Escherichia coli K12 chromosome, the EcoRI-HindIII restriction map of the section of the chromosome containing the replication origin has been extended by 14 kilobase pairs (kb) to cover 56kb. Within this newly mapped portion, the liv and rrnC cistrons have been identified by (1) hybridization of individual restriction fragmenents to the ilv-transducing phage dilv5 and (2) a comparison of the restriction map of this region with the EcoRI map of dilv5 and the HindIII map of the plasmid pJC110, a ColE1-ilv hybrid. The replication origin is located approximately 30 kb from the ilvE gene and 20 kb from the rrnC 16S rRNA cistron. This places the origin near 82.7 min on the genetic map, close to uncA.     

Acute sleep restriction alters neuroendocrine hormones and appetite in healthy male adults

This study examined the effects of two nights of sleep restriction on neuroendocrine hormones (i.e. peptide YY [PYY], ghrelin, glucagon-like peptide-1, adiponectin, and leptin) involved in regulating body weight. Ten healthy male adults aged 18 to 23 years were subjected to two consecutive nights of sleep restriction. Compared to a night of normal sleep, sleep restriction was associated with a significant reduction in PYY levels (P = 0.047) and a significant reduction in satiety levels (P = 0.033). These results suggest that sleep restriction alters the hormonal regulation of appetite in a manner predictive of increased energy intake.

     

Polymorphism in a T-cell receptor variable gene is associated with susceptibility to a juvenile rheumatoid arthritis subset

This report demonstrates a T-cell receptor (Tcr) restriction fragment length polymorphism, defined by a Tcrb-V6.1 gene probe and Bgl II restriction enzyme, to be absolutely correlated with allelic variation in the coding sequence of a Tcrb-V6.1 gene. A pair of non-conservative amino acid substitutions distinguish the Tcrb-V6.1 allelic variants. An association of this Tcrb-V6.1 gene allelic variant with one form of juvenile rheumatoid arthritis (JRA) was established in a cohort of 126 patients. The association was observed in patients possessing the HLA-DQA1*0101 gene. Among HLA-DQA*0101 individuals, 19 of 26 patients (73.1%) carried one particular Tcrb-V6.1 gene allele as opposed to 11 of 33 controls (33%; p<0.005). Haplotypes carrying this HLA gene have previously been shown to confer increased risk for progression of arthritis in JRA. This demonstration of a disease-associated Tcrb-V gene allelic variant has not, to our knowledge, been previously reported and supports the contribution of polymorphism in the Tcr variable region genomic repertoire to human autoimmune disease.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M67511 for V6.1A and M67512 for V6.1B.     

Kinetoplast DNA of Trypanosoma brucei: Physical map of the maxicircle

Trypanosoma brucei maxicircle DNA in kinetoplast DNA (kDNA) networks was characterized with restriction endonucleases. The data allow the construction of a circular map of a 22.2-kb molecule. Based on these and previous data each T. brucei kDNA network contains about 45 maxicircles which probably have the same sequence. The maxicircle of strain 164 used in this study was slightly larger and had three EcoRI sites compared to two found in other strains. Fragments generated by digestion with BamHI were largely singly cleaved maxicircles that had a density of 1.681 g/cm³ compared to 1.693 g/cm³ for the intact network. This suggests that maxicircles have a higher A + T content than minicircles. Minicircles in the kDNA network were also characterized with restriction endonucleases. Each enzyme cleaved a specific subset of minicircles from the network. However, no single restriction endonuclease or combination of up to three of these enzymes cleaved all molecules in the network. These results are consistent with earlier results of renaturation kinetic experiments and indicate that there are many different sequence classes of mini-circle DNA.     

A polymorphic system related to but genetically independent of the chicken major histocompatibility complex

Analyses of the major histocompatibility complex (Mhc) in chickens have shown inconsistencies between serologically defined haplotypes and haplotypes defined by the restriction fragment patterns of Mhc class I and class II genes in Southern hybridizations. Often more than one pattern of restriction fragments for Mhc class I and/or class II genes has been found among DNA samples collected from birds homozygous for a single serologically defined B haplotype. Such findings have been interpreted as evidence for variability within the Mhc haplotypes of chickens not detected previously with serological methods. In this study of a fully pedigreed family over three generations, the heterogeneity observed in restriction fragment patterns was found to be the result of the presence of a second, independently segregating polymorphic Mhc-like locus, designated Rfp-Y. Three alleles (haplotypes) are identified in this new system.     

Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora

Summary The organization of the ribosomal DNA (rDNA) repcat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, N. tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11+0.21 kb; standard error=0.038; coefficient of variation (C.V.)=2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5 end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.