Restriction fragment length polymorphism of the human C3 complement gene

A cloned gene-specific probe for human complement (C3) was hybridized to DNA samples digested with various restriction endonucleases. The C3 probe detects one restriction fragment length polymorphism (RFLP) that occurs frequently in the French population when DNAs are digested with Sac I. The corresponding DNA alleles can be readily used in linkage analysis of loci on chromosome 19, and such a polymorphism can be followed through myotonic dystrophy families.     

Restriction fragment length polymorphism of the DNA of typhus group rickettsiae.

The DNA of 10 strains of Rickettsia prowazekii, 5 strains of Rickettsia typhi and 1 strain of Rickettsia canada was investigated by restriction fragment length polymorphism analysis. Interspecies differences were characterized by a great number of noncomigrating bands. Using the endonuclease HindIII and PstI fragments comigration as a quantitative criterion, genetic similarity coefficient was calculated for the pair Rickettsia prowazekii/Rickettsia typhi-32.0%, for Rickettsia prowazekii/Rickettsia canada-22.7%, and for Rickettsia typhi/Rickettsia canada-23.5%. Intraspecies differences expressed are very subtle and concern 1-2 noncomigrating fragments. The investigated strains of Rickettsia prowazekii and Rickettsia typhi can be divided into 2 groups without any correlation to the source and period of isolation, or to strain passage history.     

Restriction fragment length polymorphism analysis of the V kappa locus in human lupus

To evaluate the degree of genetic polymorphism of the V kappa repertoire in systemic lupus erythematosus (SLE), we performed Southern blot hybridizations with human gene probes corresponding to the four human V kappa gene families. In a comparative analysis, non-lymphoid cell DNA samples from three patients with idiopathic SLE, eight subjects with susceptibility to drug-induced lupus and seven control individuals were digested with the restriction endonucleases Bam HI, Bg 1 II, Eco RI and Hind III, and hybridized sequentially to the four V kappa family-specific probes. The restriction patterns on Southern blots revealed a low degree of polymorphism of the human V kappa gene repertoires of SLE patients and control individuals. This analysis, together with previous parallel studies of the V kappa locus in lupus-prone mice, implies that autoantibody hyperproduction in lupus is not associated with major modifications in the structure or genomic organization of immunoglobulin light chain genes.     

Restriction fragment length polymorphism (RFLP) of a "new" HLA-DP specificity, CDP-HEI

Southern blotting with a DP beta cDNA probe of MspI digested DNA from 83 healthy unrelated individuals revealed a 1.8 kb fragment present in all four individuals (and no others) possessing the newly determined DP specificity, CDP-HEI.     

Restriction fragment length polymorphism for the identification of Campylobacter jejuni-isolates

Companion animals ("pets") are occasionally carriers of organisms pathogenic for man. In the present, study fecal samples of clinically inapparent animals with direct contact to 204 patients, suffering from campylobacter enteritis, were investigated for C. jejuni or C. coli (CJC). CJC positive animals were seen in the environment of only five patients (= 2.4%). By comparison of biotypes and serotypes of thermostable and thermolabile antigens from human and animal isolates no clear epidemiological relationship could be deduced. Using chromosomal DNA of the strains, genetic identity of the isolates was studied for HaeIII-restriction fragment length polymorphism (RFLP), applying a biotinylated commercial CJ probe. The probe was found to be specific for most CJ strains and revealed a pattern of one to four bands. In contrast to biotyping no identity of patient strains and animal isolates was seen in three cases; one case with different biotypes had identical RFLP patterns; one patient CJ strain did not show any pattern with the CJ probe. Serotypes were identical for a larger number of animal strains but differed in HaeIII RFLP and vice versa. Comparing the results from the different technological approaches it seems impossible to give a clear statement on the epidemiology of campylobacter infections or carrier state by biotyping alone. It is concluded that DNA RFLP patterns are a useful additional tool, but for epidemiological analysis a set of different methods should be used.     

Human mitochondrial DNA variation in Southern Italy

Background: Since prehistoric times Southern Italy has been a cultural crossroads of the Mediterranean basin. Genetic data on the peoples of Basilicata and Calabria are scarce and, particularly, no records on mtDNA variability have been published.

Aim: In this study mtDNA haplotypes of populations from Basilicata, Calabria and Sicily are compared with those of other Italian and Mediterranean populations, so as to investigate their genetic relationships.

Subjects and methods: A total of 341 individuals was analysed for mtDNA in order to provide their classification into haplogroups. Multivariate analysis was used to compare the studied populations with other Mediterranean samples; median-joining network analysis was applied to observe the relationship between the major lineages of the Southern Italians.

Results: The haplogroup distribution in the Southern Italian samples falls within the typical pattern of mtDNA variability of Western Eurasia. The comparison with other Mediterranean countries showed a substantial homogeneity of the area, which is probably related to the historic contact through the Mediterranean Sea.

Conclusion: The mtDNA analysis demonstrated that Southern Italy displays a typical pattern of Mediterranean basin variability, even though it appears plausible that Southern Italy was less affected by the effects of the Late Glacial Maximum, which reduced genetic diversity in Europe.     

Restriction fragment length polymorphisms of X chromosome among Japanese population

Restriction fragment length polymorphisms were studied among the Japanese using 13 polymorphic DNA probes on the X chromosome. For 6 probes (pPA4B, cpX203, p58-1, pHPGK-7e, cpX289 and 7b) the allelic frequencies were the same as those for Caucasians, but they were quite different (p less than 0.01) for 4 probes (dic56, pOTC (MspI), pTAK8B and pXG-16 (HindIII)). No polymorphisms were observed for 4 probes (pG95 alpha 1-7dIII/RI (n (chromosome number studied) = 54), pXG-16 (TaqI) (n = 50, p8 (n = 108), and pXG-17 (n = 76). These results suggest that not a small number of DNA probes currently available are useless for linkage analysis in Japan.     

Restriction fragment length polymorphism in four virulence-associated genes of Listeria monocytogenes.

We observed restriction fragment length polymorphism in 4 genes of Listeria monocytogenes associated with virulence. Using the polymerase chain reaction (PCR) and primers derived from published sequences, we amplified the following genes: hlyA coding for listeriolysin O, iap coding for a putative invasion-associated factor, mpl coding for a metalloprotease, and the prfA gene that positively regulates the hylA gene. PCR-amplified DNA were cut with several restriction endonucleases, and the restriction profiles from 29 strains, representing 6 serovars (serovars 1/2a, 1/2b, 1/2c, 3a, 3b and 4b) were compared. Based on these restriction profiles, the strains were categorized into 2 subgroups: one group contained all 10 strains of serovars 1/2a, 1/2c and 3a, the other group contained all 19 strains of serovars 1/2b, 3b and 4b. This division is in complete agreement with multilocus enzyme electrophoretic analysis data which divide the species into the same 2 subgroups. Whether the differences observed in the nucleotide sequences of the 4 virulence-associated genes for the 2 subgroups of L. monocytogenes represent salient variations in pathogenic mechanisms is not known.     

Restriction fragment length polymorphism analysis of clinical isolates of Mycobacterium haemophilum.

Mycobacterium haemophilum is an emerging opportunistic pathogen, and since 1989, infections caused by this organism have been identified more frequently in the New York City area than in any other region of the United States. A DNA fingerprinting method, based on restriction fragment length polymorphisms (RFLPs) was developed. A genomic library of M. haemophilum isolate 1A was constructed; screening the library yielded a recombinant strain that incorporated a genetic element present in multiple copies in the M. haemophilum genome. This clone was used to produce a probe for RFLP analyses of PvuII digests of genomic DNA. We used this probe to determine the RFLP patterns of 43 clinical isolates of M. haemophilum from 28 patients. A total of six distinct patterns were observed. Two patterns, designated types 1 and 2, accounted for 91% of the infections in patients from the New York City area. Two isolates from Arizona had identical patterns but were distinct from those of New York isolates, and an isolate from Israel, the type strain, had another distinct pattern (type 6). The type 6 pattern was also seen in a recent isolate from Norway. All of the type 1 isolates and 60% of the type 2 isolates were recovered from patients with AIDS in the New York City area. This molecular subtyping method should provide a useful tool for epidemiological studies and may help identify the associated risk factors, vehicles, and possible reservoirs of this newly emerging pathogen.     

Restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolated from countries in the western pacific region

A total of 422 Mycobacterium tuberculosis isolates from eight countries were subjected to IS6110 and IS1081 DNA fingerprinting by means of restriction fragment analysis to characterize M. tuberculosis strains from each country. Chinese, Mongolian, Hong Kong, Filipino, and Korean isolates had comparatively more copies of IS6110 (proportion with eight or more copies; 95% +/- 5%), while Thai, Malaysian, and Vietnamese isolates had fewer copies (proportion with eight or more copies, 60% +/- 4%). We found a number of novel IS1081 types in this study. One IS1081 type was present in 56% of Filipino isolates, had a specific 6.6-kb PvuII fragment in its IS6110 DNA fingerprint, and was termed the "Filipino family." The IS1081 types of Thai isolates had interposing characteristics between the characteristics of northeastern Asian and southeastern Asian IS1081 types. A 1.3-kb single-copy IS6110 fragment was found only in Vietnamese M. tuberculosis isolates. Although M. tuberculosis isolates from each country had comparatively similar characteristics depending on the classification factor, each country's isolates showed characteristic DNA fingerprints and differed slightly from the isolates from the other countries in either the mode number of IS6110 copies or the distribution of IS1081 types.     

Dispersed discrete length polymorphism of mitochondrial DNA in the scallop Placopecten magellanicus (Gmelin)

Three separate regions exhibiting incremental length polymorphism have been found in the mitochondrial genome of the scallop Placopecten magellanicus. Each locus has a discrete and different unit of variation, measuring 1450 bp at locus I, 250 bp at locus II, and less than 100 bp at locus III. At least six size classes were observed at each locus, and individual variation can account for the intraspecific mtDNA size range of 31–42 kb, but not for the unusually large base size of the genome. Intramolecular hybridization patterns with clones of two of the variable regions indicate that there is a dispersed sequence similarity with the 1,450-bp locus-I repeat and its flanks, and with some part of locus II.     

Restriction fragment length polymorphism analysis of ribosomal DNA intergenic regions is useful for differentiating strains of Trichophyton mentagrophytes

Twenty isolates of Tricophyton mentagrophytes var. mentagrophytes and 47 isolates of T. mentagrophytes var. interdigitale, identified by morphological characteristics, were screened by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Sixty isolates (14 of 20 T. mentagrophytes var. mentagrophytes isolates and 46 of 47 T. mentagrophytes var. interdigitale isolates) shared an identical ITS RFLP profile and were further investigated by using a probe targeted to the rDNA nontranscribed spacer (NTS) region. Polymorphisms were observed in the NTS regions of both T. mentagrophytes var. mentagrophytes and T. mentagrophytes var. interdigitale isolates. Twenty-three individual RFLP patterns (DNA types P-1 to P-12 and A-1 to A-11) were recognized and divided into two groups depending on the presence (P) or absence (A) of a 2.5-kb band, which correlated to a large extent with the morphological variety. Eleven of 14 T. metagrophytes var. mentagrophytes isolates were A types, and all of the 46 T. mentagrophytes var. interdigitale isolates were P types. A majority of strains (23 of 60 [38.3%]) were characterized by one RFLP pattern (pattern P-1), and eight types (P-1 to P-6, P-8, and P-9) accounted for 75% (45 of 60) of all strains, including all of the T. mentagrophytes var. interdigitale isolates. The remaining 15 types were represented by one only isolate and included all of the T. mentagrophytes var. mentagrophytes isolates. We conclude that RFLP analysis of the rDNA NTS region is a valuable technique for differentiation of T. mentagrophytes strains. Furthermore, by use of this method, there appears to be a greater degree of diversity among T. mentagrophytes var. mentagrophytes isolates than among T. mentagrophytes var. interdigitale isolates.     

Restriction fragment length polymorphism analysis of Cryptococcus neoformans isolates from environmental (pigeon excreta) and clinical sources in New York City.

Restriction fragment length polymorphism analysis of environmental (pigeon excreta) and clinical Cryptococcus neoformans var. neoformans isolates in a limited geographic area distinguished 6 strains among 8 environmental isolates and 12 strains among 17 clinical isolates. Clusters of patients with three strains types accounted for 47% of clinical isolates. Despite this diversity, two strains were shared by environmental and clinical isolates.     

Restriction fragment length polymorphism analysis of azole-resistant and azole-susceptible Candida albicans strains.

Restriction fragment length polymorphism analysis was performed with the endonucleases EcoRI, BglII, and HinfI on a collection of Candida albicans strains comprising eight strains randomly selected from clinical microbiology laboratory specimens, three reported azole-resistant strains from treatment failures, and several subcultures of the azole-resistant strain NCPF 3310 (also known as the Darlington strain) received from different laboratories. The results demonstrated a diversity of the restriction fragment length polymorphism patterns that were obtained and revealed that two of the proposed Darlington subcultures had patterns distinct from each other and from those of the other Darlington isolates; both were also found to have lost their azole resistance.     

Seroepidemiology of human bocavirus in Apulia,Italy

A serological survey was performed to determine the prevalence of antibodies against human bocavirus in an Apulian population. Anti-hBoV IgG antibodies were analysed in 1206 inhabitants (age range, 1 month–84 years) using a standardized ELISA test based on the use of recombinant hBoV VP2 virus-like particles. In total, 1075 (89.1%) of 1206 participants (mean age 32 ± 24.8 years) displayed anti-hBoV-IgG. The seroprevalence increased significantly (p < 0.0001) in children from 2–4 years (64.2%) to 5–9 years (96.4%). A similar trend was observed in both male and female subjects. In conclusion, our results show that hBoV infection is common in this population, especially in children.     

Restriction fragment length polymorphism analysis of 16S ribosomal DNA of Streptococcus and Enterococcus species of bovine origin.

Twelve bacterial species including Streptococcus uberis, S. parauberis, S. agalactiae, S. dysgalactiae, S. bovis, S. mitis, S. salivarius, S. saccharolyticus, Enterococcus faecium, E. faecalis, E. avium, and Aerococcus viridans were examined for their 16S ribosomal DNA fingerprint patterns. Oligonucleotide primers complementary to 16S rRNA genes were used to amplify by the polymerase chain reaction 16S ribosomal gene fragments from genomic DNAs. The molecular sizes of the amplified 16S ribosomal DNA (rDNA) fragments from the 12 species examined ranged from 1,400 to 1,500 bp. Restriction fragment length polymorphism analysis of 16S rDNA was performed with 11 different restriction endonucleases. All 12 species examined could be differentiated on the basis of characteristic 16S rDNA fingerprint patterns by using the restriction endonucleases HhaI, RsaI, and MspI. A scheme for the differentiation of the 12 species is presented. Eleven isolates representing 11 species were obtained from cows with intramammary infections and were examined by 16S rDNA fingerprinting. All 11 species isolated from cows were differentiated by using HhaI, RsaI, and MspI restriction endonucleases. The results of this study demonstrate the potential application of 16S rDNA fingerprinting for the identification and differentiation of bacterial species.     

Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus type 1 in South Africa

A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A-D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 bp fragment with AluI allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with AccI to identify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the gag p17 region (n = 33); or heteroduplex mobility assay (HMA) based on the env gene (n = 75); or both (n = 21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP gag subtypes and gag sequence subtypes demonstrating the reliability of this method. There was also no discordance between gag RFLP subtypes and env HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.     

Restriction fragment length polymorphism (RFLP) of the X chromosome linked glucose-6-phosphate dehydrogenase (G6PD) locus in India

Study: Haplotypes linked to glucose-6-phosphate dehydrogenase (G6PD) genotypes were defined by studying six intragenic restriction fragment length polymorphisms (RFLPs) in 141 G6PD deficient and 252 normal chromosomes.

Results: Only four of the 64 possible haplotypes were observed, indicating marked linkage disequilibrium. All the G6PD deficient mutations were associated with either haplotype I or VII, which are similar to the common G6PD B variant observed in the present study except the G6PD Namoru mutation which corresponded to mainly haplotype VIIa where a Nla III restriction site was created due to this mutation.

Conclusion: The limited number and low haplotype diversity probably indicates a strong selective pressure on the G6PD gene.