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The molecular diagnosis of facioscapulohumeral muscular dystrophy (FSHD) relies on detecting contractions of the unique D4Z4 repeat array at the chromosome 4q35 locus in the presence of a permissive 4q35A haplotype. Long, intact DNA molecules are required for accurate sizing of D4Z4 repeats. We validated the use of optical genome mapping to determine size and haplotype of D4Z4 alleles for FSHD analysis. The cohort included 36 unique DNA specimens from fresh blood samples or archived agarose plugs. High-molecular- weight DNA underwent sequence-specific labeling followed by separation and image analysis with data collection on the Saphyr system. D4Z4 allele sizes were calculated and haplotypes determined from the labeling patterns. Each specimen had previous diagnostic testing using restriction enzyme digests with EcoRI, EcoRI/BlnI, XapI, or HindIII, followed by pulsed field gel electrophoresis and Southern blot analysis with appropriate probes. Optical genome mapping detected 4q35 and 10q26 alleles ranging from 1 to 79 D4Z4 repeats and showed strong correlation with Southern blot allele sizing (R(2) = 0.95) and haplotyping (133 of 134; 99.4% haplotype match). Analysis of inter-assay and intra-assay runs showed high reproducibility (0.03 to 0.94 %CV). Subsequent optical genome mapping for routine clinical testing from 315 clinical FSHD cases compared favorably with historical result trends. Optical genome mapping is an accurate and highly reproducible method for chromosomal abnormalities associated with FSHD.

Background Various polymerase chain reaction (PCR)-based methods have been applied for the development of genome walking (GW) technique. These methods which could be based on the application of restriction enzymes or primers have various efficiencies to identify the unknown nucleotide sequences. The present study was conducted to design a new innovative double-strand adaptor using MAP30 gene sequence of Momordica charantia plant as a model to improve genome walking with convenient PCR. Results The adaptor was designed using multiple restriction sites of Hind III, BamH I, EcoR I, and Bgl II enzymes with no restriction site in a known sequence of the MAP30 gene. In addition, no modification was required to add phosphate, amine, or other groups to the adaptor, since restriction enzyme digestion of double-strand adaptor provided the 5′ phosphate group. Here, preparation of the phosphate group in the genomic DNA of the plant digestion with restriction enzymes was performed followed by ligation with digested adaptor containing 5′ phosphate group. Conclusion PCR was done to amplify the unknown sequence using MAP30 gene-specific primer and adaptor primer. Results confirmed the ability of the technique for successful identification of the sequence. Consequently, a newly designed adaptor in the developed technique reduced the time and cost of the method compared to the conventional genome walking; also, cloning and culturing of bacterial steps could be eliminated.

Introduction: Ischemic stroke is characterized as a sudden neurological deficit attributed to an acute focal injury of the central nervous system by a vascular cause. This study was performed to determine the frequency of G20210A mutation in the prothrombin gene and its effectiveness on the incidence of ischemic stroke in the Erbil city of Kurdistan region, Iraq. Methods: A total of 50 patients with ischemic stroke was analyzed for the detection of prothrombin gene mutation (G20210A), using polymerase chain reaction (PCR), Restriction fragment length polymorphism (RFLP) with Hind III restriction enzyme. Results: We observed no evidence of an association between ischemic stroke and G20210A mutation in the prothrombin gene in this region. Conclusion: Our finding demonstrates that prothrombotic gene variant seems not to be linked to the incidence of ischemic stroke in Erbil region.

Kappa-casein as a mammalian milk protein is involved in a several important physiological processes and it’s about 80% of the total protein in cow milk. This study aimed at genotyping bovine Kappa casein (CSN3) in two indigenous Nigerian cattle populations and to determine the frequency distribution of Kappa casein variants as detected across the animals examined and their association with the body measurement. DNA was extracted from 100 blood samples of 50 White Fulani and 50 N’dama cattle for identification and genotyping of kappa-casein gene by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) test using HindIII restriction endonucleases. The PCR products of the specific primer K-F and K-R for the two cattle breeds give 530bp specific band. Digestion of 530bp amplified products of White Fulani and N’dama by restriction endonuclease HindIII generated three fragments of 530-, 370- and 160- bp each for the two breeds. Results of the cuts with this enzyme show the presence of genotypes AA, AB and BB in the samples. These findings suggest that BB genotype could be selected for increase body conformation and protein content of milk. Olanrewaju, B. M. | Department of Animal Production and Health, The Federal University of Technology Akure (FUTA), Ondo State, Nigeria.

Cyanobacteria play a vital role in supplying nitrogen into the soil and aquatic ecosystem. It has an extra chromosomal DNA, whose role is not yet defined well. Isolation and characterization of extra chromosomal DNA in cyanobacteria might help to understand its survival mechanism. Cylindrospermum stagnale isolated (and deposited in NRMCF 3001) from soil showed presence of four plasmids namely pCYLM01, pCYLM02, pCYLM03, and pCYLM04. The following plasmids pCYLM01 and pCYLM02 were subjected to restriction digestion using HindIII restriction enzyme and cloned into pBlueScriptSK(-) vector. The sequence of pCYLM01 contained 4 potential open reading frames (ORFs) that have amino acids in the range of 59-299. Among them, ORF1 shows high sequence homology to the bacterial replication initiator family protein as evident from BLASTP analysis. The analysis of 4359 bp plasmid pCYLM02 sequence revealed 7 ORFs which are longer than 50 amino acids in length. The ORF2 of pCYLM02 has 243 amino acids and is represented in the plasmid sequence from 3045 to 3776 bp. The ORF3 of pCYLM02 corresponds to the plasmid sequence from 2323 to 2976 and codes for a putative protein of 217 amino acids long. A number of small ORFs below 50 bp were also found in the sequence analysis. Funding Agency: University of Algarve, Portugal info:eu-repo/semantics/publishedVersion

TOPARSLAN, Emine/0000-0001-8194-7842; MERCAN, Levent/0000-0002-6790-1458 WOS: 000500746600010 Thirty-five Anatolian water buffalo were selected from among a total of almost 7000 animals from 11 herds in the Kizilirmak Delta, which is known to be one of the most important areas for water buffalo farming in Turkey. The cleaved amplified polymorphic sequences (CAPS) method was used to identify single-nucleotide polymorphisms at 3 loci that are associated with milk production, namely PRL, CSN3, and PIT-1, which are associated with milk processing and milk yields, respectively. Sequence-tagged sites of PRL, CSN3, and PIT-1 were digested by restriction endonucleases RsaI, HindIII, and HinfI, respectively. Capillary electrophoretic analysis identified all samples as belonging to the GG and BB genotypes, which are undesirable genotypes for PRL and PIT-1 gene loci, but ideal for the CSN3 locus. Then the samples were sequenced by an ABI 3730 DNA Analyzer (Applied Biosystems). DNA sequencing of the amplified CSN3 fragment revealed one polymorphism at codon 135 (ThrACC > IleATC) in some samples. A homozygous structure was found for all three loci and the huge population loss between 1991 and 2008 suggests that a genetic bottleneck may have occurred in the population of Anatolian water buffalo in the Kizilirmak Delta.

The published data indicate that the bacterium Clavibacter michiganensis subsp. sepedonicus (Cms) shows a notably low degree of intraspecific variation, and most of the molecular diagnostic methods could differentiate among subspecies groups but lacked the resolution to detect genetic diversity within subspecies. The present study describes effective differentiation of 50 isolates of C. michiganensis subsp. sepedonicus by the PCR MP method using five restriction enzymes (ApaI, PstI, BamHI, XmaI, HindIII). By analysing the electrophoretic patterns obtained by the PCR MP method performed with the applied restriction enzymes, each of the 50 tested isolates could be identified and distinguished from the remaining isolates. The PCR MP method of bacterial strain identification can be used in epidemiological studies, particularly for excluding the isolates of C. michiganensis subsp. sepedonicus as a source of primary infection.

Groupers are biologically diverse, commercially important and highly regarded of all fish species associated with reef fisheries globally. Given the huge demand and high value in live fish trade, restaurants and supermarkets around the world, the accurate identification of the closely related grouper species is crucial to prevent any adulteration. Partial nucleotide sequence data of the mitochondrial cytochrome c oxidase I (COI) gene, which is globally accepted as fish DNA barcoding gene, was generated for six commercially important grouper species belonging to the genus, Epinephelus viz. E. fuscoguttatus, E. coioides, E. logispinis, E. malabaricus, E. bleekeri and E. tauvina collected from Indian waters. Based on the analysis of 704 bp COI gene sequences, a PCR-RFLP method was developed to identify all the six species accurately. The PCR products of COI gene fragment was digested with selected restriction enzymes viz. HinfI, AseI, HindIII and BsaJI. Restriction endonuclease HinfI was enabled to produce species-specific fragments for E. coioides, E. malabaricus, E. logispinis and E. bleekeri. The result shows that AseI restriction enzyme could be used to differentiate E. fuscoguttatus from other five species. The expected and actual molecular weight of the digested products was similar indicating the authenticity of the methods followed. This is the first report on differentiating closely related grouper species using above mentioned restriction endonucleases and COI gene fragment. PCR-RFLP profiles generated could be useful for detecting seafood fraud by identifying the grouper species under the genus Epinephelus using samples from fresh, frozen, cooked and even processed products.

In vivo control activities test of BCA candidate is required to evaluate its effectiveness. This test involved identification of the fungal species that inhabited the experimental plant after inoculation of BCA and pathogen. Identification of inhabitant fungi in the BCA control activity test can be conducted by culturing the fungi on artificial media. Fungal species can be identified based on morphological characters or genetic characters of fungal culture. This study was conducted to determine the potential of PCR ITS-RFLP molecular markers in the initial selection process of fungal isolates before identification based on DNA sequences and to select the enzyme that produce polymorphic PCR ITS RFLP pattern. Three enzymes (DraI, EcoRI and HinfI) were successfully separated 8 fungal cultures used in this study into three groups based on the pattern of PCR ITS DNA, while five other enzymes (BamHI, BclI HaeII, HpaI, and HindIII) were failed to cut the DNA ITS fragments, except for one isolate.

Sutarno, Zahrah S, Astirin OP, Herawati E, Setyawan AD. 2019. Genetic diversity of Ongole Grade, Aceh, and Sumbawa cattle based on polymorphism on ND-5 fragment mitochondrial DNA using PCR-RFLP technique. Biodiversitas 20: 783-788. Genetic diversity is the basis of livestock breeding because it can be used as an initial improvement in livestock quality through artificial selection. This study aims to determine polymorphism in ND-5 fragment of mitochondrial DNA in Ongole Grade, Aceh, and Sumbawa cattle and their genetic diversity. The total DNA from the blood of the local cattle was extracted using the Wizard genomic DNA purification system from Promega and amplified using the PCR technique. The PCR product was then digested with HindIII enzyme using the RFLP technique to detect polymorphism. The genetic diversity of the Ongole Grade, Aceh, and Sumbawa cattle was analyzed using the formula from Nei and its genetic relationship was evaluated with the 2.02i NTSYSpc version program. Our findings showed there were polymorphisms in the ND-5 fragment of mitochondrial DNA. Digestion with HindIII restriction enzyme produces two types of haplotypes. Haplotype B is a 453 bp-sized DNA fragment that is not truncated by the HindIII enzyme, and haplotype A is a DNA fragment cut by HindIII enzyme into two with fragments of 336 bp and 117 bp. Polymorphism was found in Ongole Grade cattle, but not in Sumbawa and Aceh cattle. Haplotypes diversity in ND-5 fragments of mitochondrial DNA of Ongole Grade was 0.6250 while Sumbawa and Aceh cattle displayed no diversity of haplotypes. The genetic relationship shows that Sumbawa cattle belonged to the same cluster with Ongole Grade but separated from Aceh cattle.

The comb is an accessory organ on the head of chicken which is influenced by testosterone hormone and can be used as an indicator of chicken’s fertility. Comb shape is related to climate adaptation and associated with a dominant mutation in chicken chromosome 1. Therefore this research was aimed to study the association between Pea-comb shape and SOX5 gene polymorphism in the population of progenies (BC-III Kambro) derived from a crossbreed between females Pelung and males second backcross generation Kampong Broiler-Type (BC-II Kambro). Chicken (Gallus gallus) SOX5 gene was acquired from NCBI GenBank with the Ref. Seq. 418195. Primers used to amplify the SOX5 gene are (F):5’-AGGTAGCCATGGTGACAAGC-3’,(R):5’-GATCTGTGAGGCAGCCAGTT-3’. Progenies showed 100% Pea-comb shape, while parental generation composed of Pea-comb shape and Single-comb shape. PCR-RFLP and endonuclease restriction enzyme HindIII were unable to determine the genotype of the female parent with Pea-comb. The result of SOX5 gene polymorphism showed the comb shape uniformity between progenies and parental of Kambro backcross generation. This study concluded that the genotype of Pea-comb shape female Pelung was undetermined and there was no polymorphism of the SOX5 gene between pea and single comb. PCR-RFLP using endonuclease restriction enzyme HindIII produced both target products and non-target/artifact products. The sequencing procedure was required to provide nucleotide sequences.

Promoter-focused chromatin conformation techniques directly detect interactions between gene promoters and distal genomic sequences, providing structural information relevant to gene regulation without the excessive non-genic architectural data generated by full scale Hi-C. 3D promoter ‘interactome’ maps are crucial for understanding how epigenomic features like histone modifications and open chromatin, or genetic variants identified in genome wide association studies (GWAS), contribute to biological function. However, variation in sensitivity between such promoter-focused methods, principally due to restriction enzyme selection, has not been systematically assessed. Here, we performed a head-to-head comparison between promoter capture C (PCC) and promoter capture Hi-C (PCHiC) with the respective 4 cutters DpnII and MboI versus 6 cutter HindIII datasets from the same five cell types. While PCHiC generally produces a higher signal to noise ratio for significant interactions in comparison to PCC, we show that DpnII/MboI detects more proximal interactions and shows little overlap with the HindIII detection range. Promoter-interacting genomic regions mapped by 4-cutters are more enriched for regulatory features and disease-associated genetic variation than 6-cutters maps, suggesting that high-resolution maps better capture gene regulatory architectures than do lower resolution approaches.

Porcine circovirus type 3 (PCV3), which currently lacks effective preventive measures, has caused tremendous economic losses to the pig husbandry. Obtaining the strain of PCV3 is the key to preparing related vaccines and developing corresponding antiviral drugs. In this study, according to the linear sequence of PCV3 DNA published on GenBank, the sequence was rearranged with SnapGene gene-editing software, and after rearrangement, the HindIII restriction endonuclease site was added to the end of the linear DNA, so that both ends have the same restriction endonuclease site. On this basis, the rearranged linear DNA is obtained by gene synthesis, PCR amplification, DNA purification, etc., and is digested and connected in vitro to obtain cyclized DNA. PCV3 infectious clones were obtained by transfecting 3D4/21 cell lines. The obtained PCV3 was identified by PCR, Western blotting, and indirect immunofluorescence tests. The results showed that this study successfully obtained the strain of PCV3 in vitro. To further evaluate the pathogenicity of the obtained PCV3 infectious clones, this study established an animal model of Kunming mice infected with PCV3. The results of RT-PCR, Western blotting and immunohistochemistry showed that PCV3 can infect myocardium and alveoli of Kunming mice, but no PCV3 was detected in other tissues. The above studies indicate that PCV3 circular DNA can be used to construct PCV3 infectious clones. This research will provide a new method for the construction of circular DNA viruses and lay the foundation for the research and pathogenesis of PCV3 vaccine.

Bovine herpesvirus 1 (BoHV-1) causes several clinical syndromes in cattle worldwide. There are 3 subtypes of BoHV-1: 1.1, 1.2a, and 1.2b. Several molecular methods are commonly used in the detection and characterization of BoHV-1. Among them, restriction endonuclease analysis (REA) and single-nucleotide polymorphism (SNP) analysis of the complete viral genome allow classification of BoHV-1 into different subtypes. However, developing countries need simpler and cheaper screening assays for routine testing. We designed a standard multiplex PCR followed by a REA assay allowing straightforward subclassification of all BoHV-1 isolates tested into 1.1, 1.2a, and 1.2b subtypes based on the analysis of fragment length polymorphism. Our standard multiplex PCR-REA was used to analyze 33 field strains of BoHV-1 isolated from various tissues. The assay confirmed the subtype identified previously by REA. In addition, non-polymorphic or undigested fragments were sequenced in order to confirm the mutation affecting the RE HindIII site. Our PCR-REA method is an affordable and rapid test that will subtype all BoHV-1 strains. Fil: Maidana, Silvina Soledad. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentina Fil: Miño, Orlando Samuel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Apóstolo, Romina María. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: De Stefano, Gabriel A.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Romera, Sonia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentina

Heterochromatin in the genome plays a vital role in species differentiation and evolution. To characterize and unravel molecular composition of the heterochromatic regions of the genome in dipteran flies, especially in Sarcophagids, different banding techniques have been used previously which established the banding pattern of constitutive heterochromatin. The present study is an attempt to further explore the base specificity of rather highly condensed C-banded regions of chromosomes using restriction enzyme (RE) digestion along with Giemsa staining, in search of species-specific targets of RE digestion. Different REs such as Apa I, Cla I, Eco RI, Hind III, Hae III and Pvu II have been used to characterize the heterochromatic regions of two Sarcophaga species, i.e., Sarcophaga ruficornis and Sarcophaga argyrostoma. Some of the constitutively heterochromatic regions remain intensely stained indicating no targets for the enzyme cleavage, while some of the heterochromatic areas in both the species show remarkable and distinct digestion sites after the treatment of REs. In S. ruficornis, Hind III digests the entire autosomes including the pericentromeric regions and the sex chromosome; while Hae III and Pvu II selectively digest autosomes and sex chromosome. While in S. argyrostoma, Cla I digests the entire autosomes including the pericentromeric regions; Hind III and Hae III selectively digests autosomes and sex chromosomes. These findings suggest the presence of distinctive enzyme digestion targets on the heterochromatin, which may provide important evidence for the differential base specificity that were previously found to be constitutive in nature.

Gurami ( Osphronemus gouramy Lac.) is a popular fish species among Indonesian people. Several Gurami strains have been cultivated by fish farmer, one of which is Gurami Soang. This strain is belived to have a faster growth rate compared to other strains. However, like other strains, the fingerling of Soang strain have also a low survival and suceptible to disease, especially that caused by A eromonas hydrophila infection. It has been proved that seeds from a single spawning event show varibale disease resistance. The difference in resistance among individuals is suggested related to the difference in their genetic component. One of the genes responsible for resistance is Major Histocompatibility Complex ( MHC ) class II B gene. Variability in resistance can be analyzed by using PCR - RFLP technique. PCR-RFLP is a technique that can produce a specific DNA fragments by PCR, followed by cutting the PCR product using restriction enzymes to describe the presence or absence of restriction sites in DNA fragments. This research aims to determine genetic marker to differiantiate between resitant and irresistant individual of Gurami Soang infected by A. hydrophila which die at a different time priod based on PCR-RFLP MHC class IIB gene. The study used survey method with purposive random sampling. The Data of PCR-RFLP band patterns were analyzed descriptively. The result indicated that cutting of the MHC class II B gene using Hinf I produce two RFLP bands with 300 bp and 100 bp length in all samples. Meanwhile, the MHC IIB gene was not cuted by Pst I, Hind III, Bam HI and Eco RI enzymes forall samples. These mean that MHC II gene in all individuals were monomorphic. Therefore,it can be concluded that there is no specific genetic marker to differentiate gurami soang individulas which was dying in different time periods.

High population of Sumateran wild boar causes the increasing number of hunting and overflowing of wild boar meat availability in the market at a very low price. This condition leads to the falsification of meat. This study was aimed to characterize and differentiate Sumateran wild boar meat (Sus scrofa vittatus) and beef (Bos taurus) since Sumateran wild boar meat is often falsified as a beef in Indonesian traditional market. The domesticated pork meat (Sus scrofa domestica) was also investigated to compare Sumateran wild boar meat and domestic pork. Samples used in this study were DNA (deoxyribonucleic acid) from pork (Sus scrofa domestica), Sumateran wild boar meat (Sus scrofa vittatus), and beef (Bos taurus). Beef and pork were obtained from traditional markets in Bogor city whereas Sumateran wild boar meat was obtained from Way Kanan Regency, Lampung Province. Herein, we performed a thorough investigation on Sumateran wild boar meat (Sus scrofa vittatus) and beef (Bos taurus) using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. This study utilized cytochrome b primer from mitochondria DNA with the size target amplification of 359 bp. This study utilized the restriction endonuclease enzymes, i.e. AluI, BsaJI, HindIII, RsaI, HaeIII, and TaqαI, in order to digest the amplification products from cytochrome b gene. The results showed that HaeIII is the merely specific enzyme to discriminate wild boar meat, domestic pork, and beef since HaeIII was able to digest these three meats in different locations of cyt b gene. However, AluI, BsaJI, HindIII, RsaI, and TaqαI enzymes were not specific enough to differentiate wild boar meat and domestic pork. In conclusion, among six tested enzymes, the use of PCR-RFLP analysis of the cyt b gene followed by digestion using HaeIII restriction enzyme provides a simple, relatively quick, and accurate identification of Sumateran wild boar species.

This study aimed to evaluate the resolution of PCR-RFLP and sequencing of trnS/trnfM in detecting genetic diversity of seagrass Enhalus acoroides at Sanur Beach, Sindhu Beach and Semawang Beach. This research used six samples E. acoroides from each location. DNA extraction method followed Doyle and Doyle. PCR amplifications were done using primers P672/P673, P674/P675 and ITS4/ITS5 followed by digestion using restriction enzymes EcoRI, PstI, HindIII, BamHI, Rsa, Mva and HinfI. Sequencing analysis used PCR products of primers trnS/trnfM. Testing of DNA sequences of E. acoroides were conducted using BLAST (Basic Local Alignment Search Tool). DNA sequences were further analyzed using MEGA 5.2 software (Molecular Evolutionary Genetic Analysis) to evaluate variations of DNA. The sequence alignments were done using ClustalW software to determine the homology between the DNA sequences. The results showed that 18 samples of E. acoroides from Sanur, Sindhu and Semawang Beach have no polymorphism based on restriction enzyme analyses. Furthermore, sequencing of trnS/trnfM region of 18 samples E. acoroides showed that the sequences were identical.

onalan, ebru/0000-0001-9968-8201 WOS: 000428836700002 The objectives of this study were to detect kappa-casein (kappa-CN, CSN3, CASK) gene polymorphisms and to investigate the association between the kappa-CNgene polymorphisms and milk yield and composition in cattle. For this purpose, a total of 365 blood and milk samples were collected from Holstein, Jersey and Brown Swiss cows. Polymorphisms of kappa-CN gene were detected by the PCR and restriction fragment length polymorphism. A776-bp fragment of exon IV was amplified by PCR and digested with HindIII and HaeIII restriction endonuclease to distinguish allele A, B and E. The analysis of associations between kappa-CN genotypes and milk yield and composition were conducted with the use of the general linear model procedure. As a result, allele frequencies were detected as 0.736, 0.357, 0.308 for A allele, 0.186, 0.643, 0.692 for B allele in Holstein, Brown Swiss and Jersey breeds, respectively, and 0.079 for E allele in Holstein. This study also demonstrated the presence of Eallele in Turkish Holstein. Although kappa-CN genotypes were associated with protein and solids-not-fat content of milk in Hostein and Brown Swiss, they were associated with milk fat content in Jerseys (P < 0.05). Scientific and Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [TOVAG-107O612] The authors gratefully acknowledge the financial support from The Scientific and Technological Research Council of Turkey (Project No: TOVAG-107O612).

We constructed a new Thermus thermophilus cloning vector which enables the colour selection of cloned DNA inserts in the T. thermophilus HB27 host strain (β-gal-) on growth plates containing 3,4-cyclohexenoesculetin β-D-galactopyranoside (S-gal) in the medium. This vector harbors a modified β-galactosidase gene (TTP0042 of T. thermophilus HB27) with 12 unique restriction enzyme sites (Acc65I, AvrII, BlpI, BssHII, EcoRI, EcoRV, HindIII, NruI, SalI, SpeI, SphI and XbaI) as multiple cloning sites under the control of the T. thermophilus slpA promoter. This host-vector system facilitates cloning procedures in T. thermophilus HB27.

Sickle cell disease shows several clinical manifestations in distinct levels of severity. This heterogeneity is due to the haplotype variability associated with the HbS gene, levels of fetal hemoglobin and environmental conditions, which modify the disease expression. Science community believes that the presence of a polymorphism in the CCR5 gene, which is related to chronic inflammatory state, could confer a higher survival rate and a lower number of inflammatory events to these patients since the deletion in CCR5Δ32 would knock out the CCR5 gene. Therefore, this study aimed to identify the haplotypes in βS and βC genes, as well as to investigate the presence of the CCR5Δ32 deletion in patients with sickle cell disease. For this purpose, DNA was isolated with the QIAamp DNA Investigator Kit, and PCR was the method chosen to detect the mutant allele CCR5Δ32. The haplotypes in βS and βC genes were detected by RFLP with the restriction enzymes XmnI, HindIII, HincII, and HinfI analyzing six polymorphic sites on the β cluster, succeeded by electrophoresis. The atypical haplotype was the most common (54.3%), followed by Benin (28.6%), Bantu (11.5%), Senegal (2.8%), and Cameroon (2.8%). No patients presented CCR5Δ32 deletion. The increase in the frequency of atypical haplotypes suggests that these patients passed by variation in the genetic pattern from ancestral haplotypes throughout the years.

Ecological damages to the Lagoon of Segara Anakan, Central Java, as well as large-scale and continuous exploitation are threatening the sustainability of fine shrimp, Metapenaeus elegans, and resources. Information in regards to genetic resources is crucial to establish long-term conservation programs and to preserve germplasm quality. This study aims to evaluate the number and size of the fragment which is digested with restriction enzyme Hind III. Seven individuals of Metapenaeus elegans from the Lagoon of Segara Anakan were examined using Hind III. Amplification of mitochondrial DNA resulted in 950 bp, and the digestion using Hind III generated four fragments consisting of 114 bp, 200 bp, 250 bp, and 386 bp, which formed a monomorphic pattern. The restriction pattern showed the probability of homozygosity of alleles that restricted using Hind III. Homozygosity indicates no variation of DNA sequence.

The study describes a rapid approach for detection of common enteric bacterial pathogens, which involves partial amplification of the 16S rRNA gene by PCR using a colony from selective medium followed by restriction enzyme (RE) digestion using the EcoRI, HindIII and SalI enzymes. On the basis of RE digestion analysis different genera namely, Escherichia, Salmonella, Shigella, Vibrio, Campylobacter, Arcobacter, Yesinia and Listeria were differentiated.

Ecofriendly synthesis of metal nanoparticles and their unique properties hold promise for innovative therapeutic applications. In the present study, rapid, novel, low cost method for synthesis of silver nanoparticles (AgNPs) using aqueous leaves extract of medicinal plant Euphorbia heterophylla ( E. heterophylla ) is reported. AgNPs synthesized by plant extract exhibited absorption peak at 422 nm in UV–vis spectroscopy. Transmission electron microscopy micrographs revealed presence of AgNPs with average size of 13 nm with grain and triangular shapes. Fourier Transform Infrared Spectroscopy analysis revealed role of leaves metabolites as reducing and capping agent in synthesis of AgNPs. X ray diffraction pattern and selected area electron diffraction study confirmed crystalline nature of AgNPs. Inhibition of DNA cutting activity of restriction endonucleases ( EcoRI, HindIII and BamHI ) by biofunctionalized AgNPs in agarose gel electrophoresis is first time reported in the present study. This potential of AgNPs can be useful for enhancing effectiveness of phage therapy by acting as synergistic cocktail with bacteriophages to kill bacteria via inhibition of their restriction endonucleases.

The 16 somatic serotype type strains and 60 field isolates of Pasteurella multocida, representing various avian species and geographic regions in Hungary, were characterised by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the ompH gene with DraI restriction endonuclease. The type strains yielded eight different (I-VIII) profiles. Strains whose PCR fragment was uncut by DraI (profile IV) could be differentiated with HindIII and PvuII restriction endonucleases. Five of the eight PCR-RFLP profiles (I, III, V, VI and VII) were detected among the field strains. Only a correlation of limited strength was found between the classical somatic serotypes and the PCR-RFLP profiles. However, the results confirmed that molecular methods could confidently distinguish serotype A:1 strains from the other serotypes. Moreover, the specific relationship between somatic serotypes and PCR-RFLP types among isolates from turkey raises the possibility of the existence of host-specific clones within the P. multocida population.

Protein and DNA polymorphismswere surveyed among seven accessions of wild fenugreek (Trigonellafoenum-graecum L.) to estimate their genetic diversity and relationships. Samples were obtained from diverse ecogeographical areas in Saudi Arabia and Yemen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of seed storage protein showed genetic variations among fenugreek germplasms, both quantitatively and qualitatively, generating a total of 168 polypeptide bands with different molecular weights ranging from 4.5 to 300 kDa. Twenty-six of these bands were polymorphic, with a considerable polymorphism value (80.00%). Furthermore, restriction fragment length polymorphism (RFLP) analysis was also employed, which was based on the ability of four restriction enzymes (EagI, EcoRI, FspI, and HindIII) to cleave genomic DNA of the plant materials at specific target nucleotide sequences into different numbers of DNA fragments. RFLP analysis revealed 166 fragments with known sequences and variable lengths ranging from 80 to 4000 bp with a highly degree of polymorphism (88.71%). Data derived from SDS-PAGE or RFLP analyses were used to produce dendrograms, which clustered the studied fenugreek accessions into different groups based on the unweighted pair group method with arithmetic mean (UPGMA). The resulting relationships indicated that these two marker techniques were nearly equivalent, but not identical, with respect to phylogenetic information. In conclusion, SDS-PAGE analysis of seed proteins should be augmented with RFLP analysis of DNA for reliable estimates of genetic diversity among fenugreek germplasms.

Two forms of the restriction enzyme HindIII were alternated with each other under some physiological or biochemical conditions. Addition of a low amount of phase T7 to the culture of HindIII-producing Haemophilus influenzae Rd, resulted in appearance of some amounts of the P2 fraction of HindIII, which was eluted with a high concentration of KCI from a phosphocellulose column. Higher amounts of T7 caused a decrease of the P2 fraction; finally the alternative PI fraction of HindIII, which was eluted with a lower concentration of KCI, remained exclusively. Addition of disaccharides such as maltose and trehalose to the bacterial extract, yielded more P2, although the disaccharides inhibited to this enzyme. Urea showed an interesting distribution of these two forms of HindIII. Phosphocellulose chromatography in the presence of 2 m urea generated a broad peak of HindIII Activity. Addition of 4 m urea, on the contrary, showed only one active peak of this enzyme. The HindIII could be purified by the following DEAE-cellulose chromatography. These results indicate the presence of only one kind of HindIII molecule, which was alternated between free and bound forms, and a certain kind of factor that would equilibrate these two forms.

Yersinia enterocolitica is currently divided into two subspecies: subsp. enterocolitica including highly pathogenic strains of biotype 1B and subsp. palearctica including nonpathogenic strains of biotype 1A and moderately pathogenic strains of biotypes 2–5. In this work, we characterized 162 Y. enterocolitica strains of biotype 1A and 50 strains of biotypes 2–4 isolated from human, animal and food samples by restriction fragment length polymorphism using the HindIII restriction enzyme. Phylogenetic relatedness of 20 representative Y. enterocolitica strains including 15 biotype 1A strains was further studied by the multilocus sequence analysis of four housekeeping genes (glnA, gyrB, recA and HSP60). In all the analyses, biotype 1A strains formed a separate genomic group, which differed from Y. enterocolitica subsp. enterocolitica and from the strains of biotypes 2–4 of Y. enterocolitica subsp. palearctica. Based on these results, biotype 1A strains considered nonpathogenic should not be included in subspecies palearctica containing pathogenic strains of biotypes 2–5. Significance and Impact of the Study Yersinia enterocolitica strains are currently divided into six biotypes and two subspecies. Strains of biotype 1A, which are phenotypically and genotypically very heterogeneous, are classified as subspecies palearctica. In this study, European Y. enterocolitica 1A strains isolated from both human and nonhuman sources were characterized using restriction fragment length polymorphism and multilocus sequence analysis. The European biotype 1A strains formed a separate group, which differed from strains belonging to subspecies enterocolitica and palearctica. This may indicate that the current division between the two subspecies is not sufficient considering the strain diversity within Y. enterocolitica.

We demonstrate that the nucleocapsid protein of Sin Nombre hantavirus (SNV-N) has a DNA-specific endonuclease activity. Upon incubation of SNV-N with DNA in the presence of magnesium or manganese, we observed DNA digestion in sequence-unspecific manner. In contrast, RNA was not affected under the same conditions. Moreover, pre-treatment of SNV-N with RNase before DNA cleavage increased the endonucleolytic activity. Structure-based protein fold prediction using known structures from the PDB database revealed that Asp residues in positions 88 and 103 of SNV-N show sequence similarity with the active site of the restriction endonuclease HindIII. Crystal structure of HindIII predicts that residues Asp93 and Asp108 are essential for coordination of the metal ions required for HindIII DNA cleavage. Therefore, we hypothesized that homologous residues in SNV-N, Asp88 and Asp103, may have a similar function. Replacing Asp88 and Asp103 by alanine led to an SNV-N protein almost completely abrogated for endonuclease activity.

Restriction endonuclease analysis (REA) typing using HindIII enzyme is a highly discriminatory, reproducible, and consistent method of genetic typing of Clostridium difficile (CD) isolates. REA typing analyzes CD whole cellular DNA on two levels of discrimination: REA Group designation and REA Type designation, which distinguishes specific subtypes within the REA Group. This methodology has enabled the tracking of epidemiologically significant CD strains over time and in some cases has allowed documentation of the evolution of previously rare REA Group strains that have subsequently become epidemic. The chapter details the methods used to isolate and purify CD colonies from stool samples, to obtain intact, full-length whole cellular DNA from CD isolates by use of guanidine-EDTA solution, and to analyze the HindIII-digested DNA after electrophoretic separation on agarose gels.

Fusarium solani is an important pathogen causing wilt disease of guava in India. In this work, we analyzed seven representative isolates of F. solani, collected from different places of India, by restriction fragment length polymorphism (RPLP) using HindIII or DraI restriction endonucleases and random amplified polymorphic DNA (RAPD). Pattern of restriction enzyme revealed a similar restriction cut type cluster in the isolate namely, Allahabad (isolate-3), Faizabad (isolate-4), Unnao (isolate-5) and Lucknow (isolate-6) region, while other cluster was consist of isolate from Ranchi (isolate-2) and Ludhiana (isolate-7). Slightly variable results were obtained when 10 randomly amplified polymorphic DNA markers (OPA01–OPA10) tested in the genome of Fusarium solani and grouped on basis of obtained allelic data. RAPD fingerprinting showed a higher variability than RFLP, and each isolate had a unique electrophoretic pattern with five of the ten primers used. Our results show that RAPD much efficient to distinguish between all F. solani isolate tested.

Sodhi, M., Mishra, B.P., Prakash, B., Kaushik, R., Singh, K.P. and Mukesh, M. 2010. Distribution of major allelic variants at exon-IV of kappa casein gene in Indian native cattle. J. Appl. Anim. Res., 38: 117–121. The present study was aimed to screen the pattern of major allelic variants of kappa casein (κ-CN) locus across 744 animals representing 17 Indian native cattle breeds adapted to different agroclimatic regions. A 935 bp fragment of κ-CN exon IV region was PCR amplified and digested with HindIII and HaeIII restriction enzymes to reveal the frequency distribution of A, B and E alleles. The digestion with HindIII pattern revealed a single 935 bp fragment or three fragments of 935, 520 and 415 bps, whereas HaeIII digestion displayed a monomorphic pattern of 641 and 294 bps fragments in all the studied samples. On analysis, only two alleles A and B were detected. AA genotype was most commonly distributed (0.818), with predominance of A allele (0.908). The present analysis indicated contrasti...

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the chloroplast DNA (cpDNA) was used to investigate phylogenetic relationships among 54 species of Salvia L. One long fragment of around 10 kilo base pair (kb) of cpDNA from Salvia was amplified using a universal pair of primers and Long Accurate-PCR (LA-PCR) protocol, then the amplified products were digested with each of six restriction enzymes; EcoRI, HindIII, PvuII, NdeI, SmaI on MfeI. Four restriction enzymes; EcoRI, HindIII, PvuII, and MfeI produced more polymorphic patterns than NdeI or SmaI. Polymorphic fragments were visually detected, documented and used to build a genetic similarity data matrix based on Nei and Li (1979) method. The genetic similarity matrix and PHYLIP software package (Felsenstein 2005) were the bases to construct a phylogenetic tree. The resulted tree divided Salvia species under study into distinct groups based on origin. Salvia roemeriana showed amplified fragment length difference (AFLD) and can be identified using any of the six enzymes. Ocimum species were distinguishable when LA-PCR products were digested with MfeI. PCR-RFLP was tried in this study due to less labor-intensive, low cost and adequate value in primary phylogenetic studies.

The present study utilizes polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using partial plastid rbcL and mitochondrial trnC–trnP gene sequences to distinguish the six representative Pyropia species produced via mariculture in Korea. The rbcL, trnC, and trnP sequences of 15 Pyropia species from the NCBI database were aligned to determine specific restriction enzyme sites of the six Pyropia species. To confirm the presence of restriction sites of eight enzymes, PCR amplicons were digested as follows: a 556 bp fragment within the rbcL region of chloroplast DNA was confirmed in P. yezoensis using BglI, whereas Tth111I, AvaII, BsrI, and BsaAI enzymes produced fragments of 664, 271, 600, and 510 bp, respectively, from the rps11–trnG region of mitochondrial DNA in P. seriata, P. dentata, P. suborbiculata, and P. haitanensis. In the case of P. pseudolinearis, HindIII, SacII, and SphI enzymes each had two cleavage sites, at positions 174 and 825, 788 and 211, and 397 and 602 bp, respectively. All six species were successfully distinguished using these eight restriction enzymes. Therefore, we propose that PCR-RFLP analysis is an efficient tool for the potential use of distinguishing between the six Pyropia species cultivated via mariculture in Korea.

An isolate of Cucumber mosaic virus (CMV), called as Lag-CMV, was identified from Lagenaria leucantha var. gourda showing mosaic symptom, and its properties was compared to Fny-CMV (subgroup IA) and As-CMV (subgroup IB) by host reaction in several indicator plants, dsRNA analysis, RT-PCR analysis, restriction enzyme profile of the PCR products and nucleotide sequence of coat protein gene. Lag-CMV was similar to As-CMV used as a control CMV by the induced chlorotic spot on inoculated leaves and mosaic symptoms on upper leaves of N. tabacum. cv. Xanthi nc. In the cucumber and zucchini squash, Lag-CMV and As-CMV induced a mild mosaic symptoms than that of Fny-CMV. Size and shapes of local lesions on Chenophodium amaranticolor and Vigna unguiculata induced by Lag-CMV was similar those by Fny-CMV or As-CMV. In experiments of dsRNA profiles and RT-PCR analysis of coat protein gene, Lag-CMV was come within subgroup I CMV. Moreover, restriction enzyme analysis using EcoRI, SalI, MspI, XhoI, and HindIII of the RTPCR products and nucleotide sequence analysis of the coat protein gene showed that Lag-CMV belong to a member of CMV subgroup IB of the same to As-CMV.

The three-dimensional crystal structures of HindIII bound to its cognate DNA with and without divalent cations were solved at 2.17 and 2.00 A resolution, respectively. HindIII forms a dimer. The structures showed that HindIII belongs to the EcoRI-like (alpha-class) subfamily of type II restriction endonucleases. The cognate DNA-complex structures revealed the specific DNA-recognition mechanism of HindIII by which it recognizes the palindromic sequence A/AGCTT. In the Mg(2+) ion-soaked structure the DNA was cleaved and two ions were bound at each active site, corresponding to the two-metal-ion mechanism.

PIRA-PCR (primer-introduced restriction analysis PCR) was developed to detect isolates of Fusarium graminearum with moderate resistance to carbendazim, a methyl benzimidazole carbamate (MBC)-group fungicide. Two primer pairs were designed and synthesized according to the nucleotide sequence of the b2-tubulin gene from F. graminearum. Fragments of 164 bp were amplified by nested PCR from isolates differing in carbendazim sensitivity. A HindIII restriction enzyme recognition site was introduced artificially by inner primers to detect a mutation at codon 167, and Taa I( Tsp4CI) restriction enzyme was used to detect a mutation at codon 200. The sensitivity of isolates to carbendazim was determined by analyzing electrophoresis patterns of the resulting PCR products after simultaneous digestion with both HindIII and TaaI. Results from PIRA-PCR and a conventional method (mycelial growth on agar) were identical but PIRA-PCR required only 7‐8 h while the conventional method required 5‐7 days. This study demonstrates that PIRA-PCR not only monitors the appearance of moderately resistant isolates, but can be useful for detecting genotypes of F. graminearum with moderate resistance to carbendazim.

Panigrahi, M., Kumar, S., Deb, S.M., Mitra, A., Sharma, A. and Bujarbaruah, K.M. 2009. Lack of polymorphism in partial insulin like growth factor 1 (IGF1) and insulin like growth factor binding protein 3 (IGFBP3) genes of mithun. J. Appl. Anim. Res., 36: 41–44. A 396 bp fragment encompassing exon 5 of Insulin like growth factor 1 (IGF1) gene and 652 bp fragment encompassing exon 2–3 of Insulin like growth factor binding protein 3 (IGFBP3) gene were digested by HindIII and NlaIII restriction enzymes, respectively, in 90 mithun (Bos frontalis). The PCR-RFLP analysis showed the absence of polymorphism in these fragments in the animals screened. HindIII restriction enzyme produced two fragments of 269 and 127 bp in exon-5 of IGF 1. Similarly NlaIII restriction enzyme produced two fragments of 551 and 101 bp in exon 2–3 ofIGFBP3 gene. Two single nucleotide differences in IGF1 while six such differences in IGFBP3 gene existed in mithun sequences when compared to cattle. The sequence of the amplicons, w...

Thirty penicillinase-producing Neisseria gonorrhoeae (PPNG) strains, 26 representing 9 serovars of the protein IB and 4 representing 2 serovars of the protein IA serogroup, had 10 different patterns when investigated by the restriction enzyme (RE) technique with Hind III enzyme. One RE pattern dominated, representing 11 strains with 6 different serovars of both the protein IA and IB serogroup. Identical serovars and RE patterns were found among 7 patients (3, 2 and 2, respectively) with positive epidemiology. The majority of the strains (24/30) were resistant to, or showed decreased sensitivity to, doxycycline with minimum inhibitory concentrations between 1 and 8 mg/l, consistent with the Far East origin. All the 30 PPNG strains showed by agarose gel electrophoresis to harbour a 2.8 Mdalton, a 4.4 Mdalton, as well as a 24.5 Mdalton plasmid, whereas the two non-PPNG strains had only the 2.8 Mdalton plasmid.

Cowpox virus (CPXV), a member of the genus Orthopoxvirus (OPV), has reservoirs in small mammals and may cause disease in humans, felidae and other animals. In this study we compared CPXVs isolated from humans and cats in Fennoscandia by restriction enzyme and DNA sequence analysis. The HindIII restriction profiles clearly distinguished geographically distinct CPXV isolates, whereas only minor differences were found between the profiles of geographically linked isolates. The complete gene sequences encoding the cytokine response modifier B, the hemagglutinin and the Chinese hamster ovary host range protein were determined for the same isolates and included in phylogenetic analysis. By including representative OPV sequences from GenBank, detailed comparative analyses were performed showing pronounced heterogeneity among CPXVs compared to members of other OPV species. However, a close relationship between the Norwegian (3 of 4 isolates) and Swedish isolates was detected, whereas the isolate from Finland was more closely related to a Russian isolate for all three genes compared. We infer that the investigated CPXVs have distinct evolutionary histories in different rodent lineages.

The present work is undertaken mainly for genotyping of kappa-casein gene in buffalo bulls. DNA was extracted from the frozen semen of nine buffalo bulls used in artificial insemination in addition to 40 blood samples of male buffalo calves for identification and genotyping of kappa-casein gene by PCR-RFLP assay. Different restriction endonucleases, including HindIII, HinfI and Taq1 were used. The PCR product of the primer specific for K2 and KY gives the specific band at size 379 and 280 bp respectively. Digestion of 379 bp fragment K2 by restriction endonuclease HindIII generated two fragments of 156 and 223 bp. For fragment 280 bp kappa-casein KY, the above mentioned endonuclease generated two fragments of 180 and 100 bp. Digestion of the 379 bp PCR product with HinfI resulted two restriction fragments of 288 and 91 bp. On the other hand, the amplified DNA (379 bp) from buffalo remained undigested by Taq1 restriction enzyme. In conclusion, the PCR-RFLP technique demonstrated that bulls were monomorphic for the kappa-casein gene; they possess only allele B in homozygosis form.

Restriction fragment length polymorphism was studied in an ELA typed horse family which included a stallion, a mare with two full-sibs, another mare with three full-sibs and, in addition, three paternal half-sibs. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI) and human cDNA class I, class II (DR beta) and class III (C4) probes. In addition, a genomic class II DQ alpha probe was used. Fragments hybridized with the various probes revealed the existence of DNA sequences homologous to HLA class I, DR beta, DQ alpha and C4 genes in the horse. Polymorphic fragments were found when DNA was hybridized with class I and class II probes irrespective of the enzyme used; but hybridization with the C4 probe did not reveal variability. All polymorphic fragments segregated according to the ELA serological specificities, thus indicating a close linkage between the different revealed subregions. Banding patterns suggest that the horse possesses about 20-30 class I genes, probably more than one DR beta and DQ alpha genes and possibly only one C4 gene. The high degree of polymorphism observed suggests that molecular DNA typing may represent a potentially powerful aid to decision in parentage control determination.

Restriction fragment length polymorphism (RFLP) of ovine casein genes was investigated. Genomic DNA from 56 rams was digested with 10 restriction endonucleases and Southern blots probed with the four ovine casein cDNAs (alpha s1-, beta-, alpha s2- and kappa-Cn). Five enzymes, namely, BglI, PvuII, RsaI, TaqI and HindIII revealed nine different RFLPs. The inheritance of six of these polymorphisms was studied by segregation analysis of gametes in nine rams' families, and each of them could be related to the existence of alleles at the relevant casein locus. A close linkage between the four ovine casein genes was demonstrated since no recombination within the four pairs of loci examined, alpha s1-beta-Cn, alpha s1-kappa-Cn, beta-kappa-Cn and alpha s2-kappa-Cn, was observed in the progeny of double heterozygous rams. The casein genes are thus clustered in the ovine species as in the case of other mammals.

High molecular weight DNA was extracted from sperm from chickens of 14 inbred lines. The DNA was digested with each of four restriction enzymes (Pvu II, Hind III, Bgl II, and Bam HI), electrophoresed for 18 or 45 h, blotted onto nitrocellulose, and hybridized to a chicken major histocompatibility complex (MHC, B complex) class II beta-chain probe (beta 2-exon specific). Restriction fragment length polymorphisms (RFLPs) were found with each of the restriction enzymes used. Birds with the same B haplotype always showed the same RFLP pattern; however, some birds of different B haplotypes also shared the same RFLP pattern. To test for the Mendelian inheritance of the RFLP patterns, the F2 progeny of an informative cross were analysed. The RFLP patterns corresponded with the serologically determined B haplotypes of the F2 birds, thereby showing the Mendelian inheritance of the polymorphic bands.

An isolate of Cucumber mosaic virus (CMV), designated as Cas-CMV, was isolated from Chinese aster (Callistephus chinensis) showing severe mosaic symptom, and its properties was compared to the well-characterized Fny-CMV (subgroup IA) and As-CMV (subgroup IB) by host reaction in several indicator plants, dsRNA analysis, RT-PCR analysis, and restriction enzyme profile of the PCR products. Cas-CMV differed markedly in their host reaction to Fny-CMV or As-CMV in Cucurbita pepo cv. Black beauty. In the zucchini squash, all strains induced chlorotic spot on inoculated leaves and mosaic symptoms on upper leaves. However, symptoms induced by Cas-CMV were developed lethal necrosis on the young plants 15 to 20 days after inoculation. In experiments of dsRNA analysis and RT-PCR analysis, properties of Cas-CMV was come within subgroup I CMV. Moreover, restriction enzyme analysis using HindIII of the RT-PCR products showed that Cas-CMV belong to a member of CMV subgroup IA.

To study the association of renin gene polymorphism with essential hypertension in the Chinese population, 86 hypertensive and 107 normotensive subjects were enrolled from an epidemiologic survey. Leukocyte DNA was extracted and digested with Hind III and Bgl I restriction enzymes. Southern hybridization was done with digoxigenin-incorporated renin gene probes generated by polymerase chain reaction. The restriction fragments were detected by anti-digoxigenin antibody and enzyme methods. Two Hind III polymorphysms of the renin gene (8.7 kb and 6.2 kb) were identified. The allele frequences were 129(75%) and 43(25%), respectively, in hypertensives; they were 139(65%) and 75(35%), respectively, in normotensives (chi2 = 4.074, p = 0.044). The genotypes of 8.7/8.7,8.7/6.2 and 6.2/6.2 were significantly different between hypertensives and normotensives, being 45(52%), 39(45%), 2(3%) and 48(45%), 43(40%), and 16(15%), respectively (chi2 = 9.002, p = 0.11). The Bgl I polymorphism did not show a difference between hypertensives and normotensives. Thus, we conclude that the renin gene Hind III polymorphysm is associated with hypertension in this Chinese population.

Dogru, U., Ozdemir, M. and Ercisli, S. 2008. Genetic polymorphism in Kappa-casein gene detected by PCR-RFLP in cattle. J. Appl. Anim. Res., 33: 65–68. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method has been used to detect polymorphism in Kappa-casein gene in cattle. A 351 bp Kappa-casein gene fragment was amplified and the PCR products were digested with four different restriction enzymes i.e. Alu I, Hind III, Hinf I and Taq I. All these enzymes were able to distinguish between Kappa-casein A and B types, however Hind III, Hinf I and Taq I were found more effective for kappa-casein variants than Alu I.

An improved method to identify the T-DNA insertion site in transgenic Arabidopsis thaliana (Columbia ecotype) genome was presented. Firstly, the pre-adaptor was amplified by PCR from the plasmid pLASC11.12.8 and digested by HindIII to produce the adaptor. After treated with calf intestine alkaline phosphatase, the adaptor was ligated to the genomic restriction digested fragment with the same restriction endonucleases. Then two rounds of PCR (nested-PCR) were carried out and an unknown sequence between the T-DNA and the adaptor was amplified. Further analysis would reveal the accurate site of T-DNA insertion into transgenic A. thaliana genome.

A salt tolerant gene was isolated and characterized from a highly salt tolerant soil - borne bacterium, Bacillus coagulans. The genomic library was prepared in ZAP expression vector. The amplified library had inserts ranging from 0.5–3.8 kb. The library was screened for isolation of salt tolerant recombinant clones by functional assay using LB agar containing 4% NaCl. Salt tolerant recombinant clone was able to grow much faster than the wild strain on LB agar plates containing 4% NaCl. Size of the gene responsible for salt tolerance was 3.8 kb and had restriction sites for a number of restriction enzymes like HindIII, SmaI, PstI, ClaI, SacI and SpeI. The restriction map of the gene was developed through single and double digestion with the restriction enzymes. The sequence of the insert revealed ORF of 393 amino acids and using BLASTP nr-na database, the insert was identified as ornithine amino transferase, which is known to play an important role in biosynthesis of proline — an important osmolyte involved in salt tolerance of many species of bacteria and plants.

One hundred and seventy-six Enterococcus faecium isolates from Slovak dairy product Bryndza were tested for the presence of plasmid DNA. Eighty-two isolates were positive and their plasmid DNA was isolated and digested by EcoRI and HindIII restriction endonucleases. The patterns obtained were compared with those obtained after pulsed-field gel electrophoresis of macrorestriction fragments (PFGE), (GTG)(5)-PCR and ERIC-PCR. All these molecular approaches were applied for the study of genetic variability and determination of strain relatednesses among plasmid-positive isolates of E. faecium. In general, all methods revealed a considerable genetic diversity of E. faecium isolates. Plasmid profiling and ERIC-PCR have offered a higher resolution than PFGE and (GTG)(5)-PCR.