Evolutionary relationships between laboratory mice and subspecies of Mus musculus based on the genetic study of pancreatic proteinase loci,Prt-1, Prt-2, Prt-3, and Prt-6

Various patterns of mouse pancreatic proteinase activity bands were observed on agarose gel electrophoresis. Prt-1 ^a and Prt-1 ^b genes control the positive (PRT-1A) and negative (PRT-1B) expression of tryptic band V, respectively; Prt-2 ^a and Prt-2 ^b correspond to chymotryptic bands II (PRT-2A) and III (PRT-2B); Prt-3 ^a and Prt-3 ^b control the low (PRT-3A) and high (PRT-3B) tryptic activities of band IV; the Prt-1 and Prt-3 loci are closely linked on the same chromosome; Prt-6 ^a and Prt-6 ^b correspond to tryptic bands I (PRT-6A) and I (PRT-6B). Twenty-four laboratory strains from the United States showed the phenotype PRT-1A, PRT-3A, and PRT-2A. Of laboratory strains established in Europe, 6 showed PRT-1A, PRT-3A, and PRT-2A, and 10 had PRT-1B, PRT-3A, and PRT-2A bands. Most wild mice around the world and their descendants showed the phenotype PRT-1B, PRT-3B, and PRT-2A. Only the phenotype of M. m. brevirostris was PRT-1A, PRT-3A, and PRT-2A, which was the same as most laboratory inbred strains. PRT-2B was observed mainly in Japanese (M. m. molossinus) and Korean (M. m. yamashinai) wild mice. PRT-6B was detected only in Mus spicilegus and Mus caroli, but all other mice including wild populations and laboratory strains showed PRT-6A. New biochemical phenotypes such as PRT-2C and PRT-3C were also found in this study.     

Mapping of theHox-3.1 andMyc-1.2 genes on chromosome 15 of the mouse by restriction fragment length variations

Restriction endonuclease fragment length variations (RFLV) were detected by use of the cDNA probeHox-3.1 for the homeo box-3.1 gene and also thec-myc oncogene probe for exon 2. RFLV ofHox-3.1 were found inHindIII restriction patterns, and RFLV of theMyc-1.2 gene inEcoRV patterns. From the RFLV, theHox-3.1 andMyc-1.2 genes were mapped on chromosome 15. Three-point cross test data showed that the frequency of recombination is 26.4% betweenMyc-1.2 andGpt-1, 30.2% betweenGpt-1 andGdc-1, and 9.4% betweenGdc-1 andHox-3.1. The following order of these genes is proposed,Myc-1.2—Gpt-1—Gdc-1—Hox-3.1. All laboratory strains carry theHox-3.1 ^a andMyc-1.2 ^a alleles. Among strains of wild origin,domesticus strains carry only theHox-3.1 ^a andMyc-1.2 ^a alleles, as do the laboratory strains. One strain ofbrevirostris carries theHox-3.1 ^a andMyc-1.2 ^b alleles. Other wild subspecies from Europe and Asia,M. m. musculus, M. m. castaneus, M. m. molossinus, Chinese mice of wild origin, andM. m. yamashinai carry theHox-3.1 ^b andMyc-1.2 ^b alleles.     

Restriction fragment length variants in the marsupialSminthopsis crassicaudata

A fully pedigreed colony of the dasyurid marsupialSminthopsis crassicaudata has provided material for establishing two panels of DNA samples: a broad-based test panel and a two-generation family panel. These have been used to search for genetic markers in the form of restriction fragment length variants. The molecular probes—pSG-2H, a region of theS. crassicaudata embryonic -globin gene; pB8.BS, a region of the human ubiquitin gene, and p3-21a1:1, a region of the processed pseudogene of phosphoglycerate kinase-1 of the macropodid marsupialMacropus robustus—were hybridized to Southern blots ofEcoR1-digested DNA from the panels. Analysis of these blots when probed with pSG-2H provided evidence of two alleles segregating at a singleEcoR1 site. Analysis of the same blots when probed with pB8.BS suggested allelic variation at two closely linkedEcoR1 sites. Probing the blots with p3-21a1:1 produced a complex pattern of bands resembling DNA fingerprints. The presence of a 12.3-kb band was found to conform to a simple autosomal dominant mode of inheritance. Analysis of the family data, for each probe, revealed no significant departure from Mendelian inheritance. This work has provided additional genetic markers that will enhance the use ofS. crassicaudata as a model marsupial species and has demonstrated that a high level of genetic variability has been maintained in the marsupial colony.This project was supported by grants from the Australian Research Council and the University of Adelaide.     

Comparing the frequencies of restriction fragment length polymorphisms for dystrophin gene in Chinese with those from Japanese and Caucasian populations

The restriction fragment length polymorphisms distribution and frequency of dystrophin gene in Chinese were studied by using 14 subclones of the entire 14kb cDNA for the dystrophin as hybridization probes. Allelic fragments were detected in hybridization patterns of PvuⅡ/la, Taq Ⅰ/2b-3, Taq Ⅰ/5b-7, and Xba Ⅰ/10. Among them, the allelic fragments (26kb and 3.8kb) in PvuⅡ/2b-3 pattern and the allelic fragments (10.0kb and 8.4kb) in Taq Ⅰ/5b-7 patterns had never been reported previously. Compared with the data from Caucasians and Japanese, it indicated that there was a significant difference (P<0.01) of the allelic fragment frequency in Taq Ⅰ/2b-3 and Xba Ⅰ/10 patterns between Chinese and Caucasians. The frequencies of allelic fragments A2 (5.6kb) in Taq Ⅰ/8 and A2 (10.Tkb) in EcoR Ⅴ/9 were high in Caucasians, yet had not been detected in Chinese, the differences were also highly significant. But in Chinese and Caucasians, the B1B2 allelic frequencies in Taq Ⅰ/5b-7 are the same. As to the frequency of the allelic fragments A1A2 and B1B2 in Pvu Ⅱ/la, there was no significant difference between Chinese and Japanese.     

Differentiation of the endemic New Zealand greenheaded and brownheaded leafroller moths by restriction fragment length variation in the ribosomal gene complex

This study identified markers in the ribosomal RNA gene complex (rDNA) and evaluated their utility in the differentiation of four endemic New Zealand species of leafroller moth, Planotortrix excessana (Walker), P. octo (Dugdale), Ctenopseustis herana (Felder and Rogenhofer) and C. obliquana (Walker). Pooled genomic DNA from laboratory-reared specimens of C. herana and C. obliquana could be distinguished with a single restriction enzyme (e.g. ClaI), while P. excessana and P. octo could be satisfactorily distinguished using double digestions with either ClaI/PvuII or ClaI/BglII. It is concluded mat these markers may be used to identify moths from wild populations.     

Genetic variability between two breeds based on restriction fragment length polymorphisms (RFLPs) of major histocompatibility complex class I genes in the pig

Summary Restriction fragment length polymorphism analyses of SLA class I genes were performed on 55 Duroc and 24 Hampshire boars from the 1986–87 national performance tests of each breed. Few boars were inbred. Southern blotting and hybridization procedures were performed on genomic DNA isolated from white blood cells by using Pvu II, Bam HI, and Eco RI endonucleases and a swine MHC class I probe. Genetic variability within and between the two breeds was estimated in terms of nucleotide diversity, by using a mathematical analysis based on the different RFLP patterns. The nucleotide diversity calculated within each breed was less than that between the two breeds. The results from the nucleotide diversity analysis suggested that genetic variability was greater in the Duroc breed than in the Hampshire breed. A relatively high level of genetic variability was shown in the class I major histocompatibility complex genes in the pig.     

Association of restriction fragment length polymorphisms of swine leucocyte antigen class I genes with production traits of Duroc and Hampshire boars

Summary. Restriction fragment length polymorphism analyses of swine leucocyte antigen (SLA) class I genes were performed on 70 Duroc and 38 Hampshire boars from the 1986-87 national performance tests of each breed in the USA. Few boars were inbred. Southern blotting and hybridization procedures were performed on genomic DNA, isolated from white blood cells, using Pvu II endonuclease and a swine major histocompatibility complex (MHC) class I probe. Durocs had an average of 11 restriction fragments, with the most common being in 63% of the boars and the least common appearing in only one boar. Hampshire boars had an average of 12 restriction fragments, with the most common appearing in 73% of the boars and the least common appearing in only one boar. Least squares procedures and stepwise regression methods were used to examine the association between DNA restriction fragments and the selection index (INDEX), average daily gain (ADG), average backfat thickness (BF), loin muscle area (LEA), and age at 104kg (DAY104). In the Duroc breed one DNA restriction fragment was associated with decreased INDEX ( P < 0.05) and decreased ADG ( P < 0.05) whereas two other fragments were associated with increased BF ( P < 0.05). In the Hampshire breed two restriction fragments were associated with an increase in INDEX ( P < 0.05). Cluster analyses were used to group pigs of each breed on the basis of similar RFLP patterns. One cluster group in the Duroc breed was associated with lower average INDEX values ( P < 0.05), greater average DAY104 ( P < 0.05), and a larger mean LEA ( P < 0.05). In the Hampshire breed one cluster group was associated with lower INDEX ( P < 0.05). These results suggest there may be an association between swine MHC class I genes and performance traits in swine. The use of SLA class I restriction fragments, as genetic markers, may have potential in the future for improving pig performance.     

Comparison of two commercial methods with PCR restriction fragment length polymorphism of the tuf gene in the identification of coagulase-negative staphylococci

AIMS: Two commercial methods for the identification of coagulase-negative staphylococci (CNS) were compared with the restriction fragment length polymorphism (RFLP) of the amplified tuf gene, which served as the reference method. METHODS AND RESULTS: One hundred and forty-five CNS were evaluated using the API 32 Staph ID and the Crystal GP/ID BBL systems. The PCR-RFLP of the tuf gene served as the reference method. The APIStaph and the GP/ID BBL had an overall rate of agreement with the molecular method of 58.6% and 46.2% respectively, with the inability of the GP/ID BBL to characterize 11.7% of the isolates. The APIStaph showed higher sensitivity and better agreement than the GP/ID BBL with the PCR-RFLP, except for Staphylococcus hominis and Staphylococcus capitis. CONCLUSIONS: Neither of the commercial systems was as reliable as the PCR-RFLP method for identifying isolates of CNS. Overall the APIStaph had better agreement with the PCR-RFLP than the GP/ID system. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the PCR-RFLP method is more reliable than the two commercial systems tested, suggesting that it is more reliable for routinely identifying CNS.     

Use of restriction fragment length polymorphisms to identify sea turtle eggs and cooked meats to species

One of the many threats to sea turtlepopulations is the take of turtles and theireggs for consumption and sale. Improved speciesidentification methods for sea turtle eggs andcooked meats would facilitate prosecution ofthose involved. Fatty acid-based methods toidentify eggs cannot resolve loggerheads andthe two ridley species. Protein-based methodsare not applicable to eggs or cooked meat. Wepresent methods to extract DNA from turtle eggand cooked meat and to produce diagnosticrestriction fragment length polymorphismpatterns in the cytochrome b region of themitochondrial DNA. This method works on DNAfrom any tissue, and provides wildlife lawenforcement another tool to combat illegal takeof endangered species.     

Detection of linkage between quantitative trait loci and restriction fragment length polymorphisms using inbred lines

Summary In segregating populations, large numbers of individuals are needed to detect linkage between markers, such as restriction fragment length polymorphisms (RFLPs), and quantitative trait loci (QTL), limiting the potential use of such markers for detecting linkage. Fewer individuals from inbred lines are needed to detect linkage. Simulation data were used to test the utility of two methods to detect linkage: maximum likelihood and comparison of marker genotype means. When there is tight linkage, the two methods have similar power, but when there is loose linkage, maximum likelihood is much more powerful. Once inbred lines have been established, they can be screened rapidly to detect QTL for several traits simultaneously. If there is sufficient coverage of the genome with RFLPs, several QTL for each trait may be detected.     

Identification of Azospirillum strains by restriction fragment length polymorphism of the 16S rDNA and of the histidine operon

Abstract DNA fingerprints of several Azospirillum strains, belonging to the five known species A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum , were obtained by restriction analysis of the amplified 16S rDNA and by restriction fragment length polymorphism of the histidine biosynthetic genes. Data obtained showed that amplified rDNA restriction analysis is an easy, fast, reproducible and reliable tool for identification of Azospirillum strains, mainly at the species level, whereas restriction fragment length polymorphism could, in some cases, differentiate strains belonging to the same species. Moreover, both analyses gave congruent results in grouping strains and in the assignment of new strains to a given species.     

Genetic diversity of oilseedBrassica napus germ plasm based on restriction fragment length polymorphisms

Oilseed rape (Brassica napus) is an important oilseed crop worldwide. Cultivars have been developed for many growing regions, however little is known about genetic diversity inB. napus germ plasm. The purpose of the research presented here was to study the genetic diversity and relationships ofB. napus accessions using restriction fragment length polymorphisms (RFLPs). Eighty threeB. napus accessions were screened using 43 genomic DNA clones which revealed 161 polymorphic fragments. Each accession was uniquely identified by the markers with the exception of the near-isogenic cvs Triton and Tower. The RFLP data were analyzed by cluster analysis of similarity coefficients and by principal component analysis. Overall, there were three major groups of cultivars. The first group included only spring accessions, the second mostly winter accessions and the third, rutabagas and oilseed rape accessions from China and Japan. These results indicate that withinB. napus, winter and spring cultivars represent genetically distinct groups. The grouping of accessions by cluster analysis was generally consistent with known pedigrees. This consistency included the grouping of lines derived both by backcrossing or self-pollination with their parents.     

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri , Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii . Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.     

Isolation of cDNAs for mouse phenol and bilirubin UDP-glucuronosyltransferases and mapping of the mouse gene for phenol UDP-glucuronosyltransferase (Ugtla1) to chromosome 1 by restriction fragment length variations

The mouse gene for phenol UDP-glucuronosyltranferase (UDPGT;Ugtla1) was mapped at 42 cM on chromosome 1, a position identical to that of the gene for bilirubin UDPGT (Ugtla1), from linkage analysis of a three-point cross test withIdh-1, En-1, andUgtlal as marker genes. The cDNAs for mouse phenol and bilirubin UDPGTs, isolated after amplification by PCR, shared an identical 3-half region. Our results strongly suggest that mouse bilirubin and phenol UDPGTs are expressed from a single gene and involve alternative splicing events. We also detected duplication of the gene for phenol UDPGT in all mouse strains examined with the exception of MOL-MIT and SUB-SHH.     

Characterisation of the lpdA gene from Neisseria meningitidis by polymerase chain reaction, restriction fragment length polymorphism and sequencing

P64k protein from Neisseria meningitidis is well recognised in sera from individuals convalescent from meningococcal disease or vaccinated with the Cuban antimeningococcal vaccine VA-MENGOC-BC. The presence of the protein in more than 80 meningococcal strains has also been verified. It is immunogenic in animal models and the antibodies elicited show bactericidal activity against meningococci. To further investigate at the molecular level whether lpdA, the gene coding for P64k protein, is conserved among different N. meningitidis strains, a total of 20 strains isolated from different geographic areas were differentiated on the basis of restriction fragment length polymorphism (RFLP) patterns after polymerase chain reaction (PCR) amplification of the lpdA gene and restriction endonuclease digestion with HpaII. Although a total of five different PCR-RFLP patterns were present, nucleotide sequence determination showed that identity levels were as high as 93-99% among the N. meningitidis strains analysed.     

Restriction fragment length polymorphisms in dairy and beef cattle at the growth hormone and prolactin loci

Two bovine populations, a Holstein-Friesian dairy stock and a synthetic (Baladi X Hereford X Simmental X Charolais) beef stock, were screened for restriction fragment length polymorphisms (RFLPs) at the growth hormone and prolactin genes. Most RFLPs at the growth hormone gene are apparently the consequence of an insertion/deletion event which was localized to a region downstream of the structural gene. The restriction map for the genomic region including the growth hormone gene was extended. Two HindIII RFLPs at the growth hormone locus, as well as several RFLPs at the prolactin gene, seemed to be the consequence of a series of point mutations. The results are discussed in terms of the possibility that minor genomic variability underlies quantitative genetic variation.     

Restriction fragment length polymorphism of DQB and DRB class II genes of the ovine major histocompatibility complex

The ovine major histocompatibility complex (MhcOvar) class II region was investigated by Southern blot hybridizations using ovine probes specific for the second exons of Ovar-DRB and Ovar-DQB genes. Multiple bands were revealed when genomic DNA was digested with each of five restriction enzymes (Bam HI, Eco RI, Hin dIII, PvuII and TaqI), and successively hybridized with the two radiolabeled ovine probes. Restriction fragment length polymorphisms (RFLPs) were analysed in 89 sheep originating from six inbred families and the inheritance of the fragment patterns was determined. Forty-one fragments were recorded with the DQB probe; 32 were detected with the DRB probe. They constituted 9 DQB and 10 DRB allelic patterns. Twelve DQB-DRB haplotypes were resolved in this study.     

Mitochondrial restriction fragment length polymorphism (RFLP) and sequence variation among closely related avian species and the genetic characterization of hybrid Dendroica warblers

To address several interconnected goals, we used mitochondrial DNA (mtDNA) sequences to explore evolutionary relationships among four potentially hybridizing taxa in a North American avian superspecies (Dendroica occidentalis, D. townsendi, D. virens, and D. nigrescens). We first compared the results of a previous restriction fragment length polymorphism (RFLP)-based study with 1453 nucleotides from the mitochondrial cytochrome oxidase subunit I (COI), ATP-synthase 6 (ATPase 6), and ATP-synthase 8 (ATPase 8) genes. Separate phylogenetic analyses of the RFLP and sequence data provided identical and well-supported hierarchical species-level reconstructions that grouped occidentalis and townsendi as sister taxa. We then explored several general features of mitochondrial evolution via a comparison of the RFLP and sequence data sets. Qualitative rate differences that seemed evident in highly autocorrelated comparisons of RFLP vs. sequence pairwise distances were not supported when autocorrelation was removed. We also noted a high variance in corresponding RFLP and sequence distances after the removal of autocorrelation effects. This variance suggests that caution should be used when combining RFLP and sequence-based data in studies that require the large-scale synthesis of divergence estimates drawn from sources employing different molecular techniques. Finally, we used our parallel RFLP and sequence data to design and validate a rapid and inexpensive polymerase chain reaction-RFLP (PCR-RFLP) protocol for determining species-specific mitochondrial haplotypes. This PCR-RFLP technique will be applied in ongoing studies of the occidentalis/townsendi hybrid zone, where the historic and geographical complexity of the interbreeding populations necessitates the genotyping of thousands of individual warblers.