Genomic DNA Blot Hybridization Research Articles

Prevalence and molecular characteristics of mcr-1 gene in Salmonella typhimurium in a tertiary hospital of Zhejiang Province.

Objectivesmcr-1 gene has been widely reported in the world. This study aimed to analyze the prevalence and molecular characteristics of mcr-1 gene in Salmonella typhimurium from Quzhou People’s Hospital.Materials and methodsA total of 62 S. typhimurium isolates were isolated and preserved in our laboratory from 2007 to 2016. PCR was used to screen plasmid-mediated colistin resistance gene, mcr-1. For mcr-1-positive isolates, susceptibilities to colistin and other antibiotics were assessed using broth microdilution or agar dilution methods. The genetic location of mcr-1 was determined by analysis of pulsed-field gel electrophoresis profiles of S1-digested genomic DNA and subsequent Southern blot hybridization. The multi-locus sequence type and other drug resistance genes found in the mcr-1-positive isolates were analyzed by performing whole genome sequencing. Genetic environment of mcr-1 gene was analyzed by RAST and Easyfig.ResultsA total of three S. typhimurium isolates were identified to be mcr-1 positive, with the prevalence rate of 4.8% (3/62). The minimum inhibitory concentration values of colistin for all these isolates were 8 µg/mL. The three mcr-1-positive isolates carried mcr-1 gene on two different types of plasmids having the sizes of ~54.7–78.2 kb and 310.1 kb, respectively. All the three isolates belonged to ST34 and carried various resistant genes.ConclusionColistin-resistant, mcr-1-positive S. typhimurium isolates belonging to ST34 have been isolated from Quzhou People’s Hospital. Surveillance needs to be strengthened to identify colistin resistance and prevent the spread of drug-resistant bacteria in the hospital.

Lymantria mathura nucleopolyhedrovirus: Identification, occurrence and genetic diversity in Iwate Prefecture, Japan

A high prevalence of nuclear polyhedrosis has been observed in larval outbreaks of Lymantria mathura in Iwate Prefecture, Japan. However, the virus responsible has not been identified. Here the virus was designated Lymantria mathura nucleopolyhedrovirus (LymaNPV), based on partial sequence results of the lef-8, lef-9, and polh genes and transmission electron microscopic observations. Diagnosis by polymerase chain reaction targeting of the partial polh gene and a subsequent restriction fragment length polymorphism (RFLP) analysis indicated that LymaNPV was an exclusive causative agent for the nuclear polyhedrosis of the L. mathura larvae. LymaNPV was also detected from a very small fraction of L. dispar larvae that co-occurred with L. mathura larvae where the prevalence of LymaNPV-caused disease was high. A bioassay using LymaNPV against L. dispar larvae produced infection with the inoculated virus. This finding was consistent with the results of the field sample analysis. LymaNPV contained six genome types in the L. mathura populations sampled, as determined by RFLP of LymaNPV genomic DNA and southern blot hybridization analyses. None of the genome types was unique to any sampling site, indicating that some flow had occurred among sites. However, genome-type composition seemed to differ among sites. This study provides basic information about the interaction between L. mathura and LymaNPV.

Horizontal Acquisition of a Multidrug-Resistance Module (R-type ASSuT) Is Responsible for the Monophasic Phenotype in a Widespread Clone of Salmonella Serovar 4,[5],12:i:.

Salmonella enterica serovar 4,[5],12:i:- is a monophasic variant of S. Typhimurium incapable of expressing the second-phase flagellar antigen (fljAB operon), and it is recognized to be one of the most prevalent serovars causing human infections. A clonal lineage characterized by phage type DT193, PulseNet PFGE profile STYMXB.0131 and multidrug resistance to ampicillin, streptomycin, sulphonamides and tetracycline (R-type ASSuT) is commonly circulating in Europe. In this study we determined the deletions affecting the fljAB operon and the resistance region responsible for the R-type ASSuT in a strain of Salmonella enterica serovar 4,5,12:i:- DT193/STYMXB.0131, through an approach based on PCRs and Southern blot hybridization of genomic DNA. Using a set of nine specific PCRs, the prevalence of the resistance region was assessed in a collection of 144 S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 strains isolated from Germany, Switzerland and Italy. A 28 kb-region is embedded between the loci STM2759 and iroB, replacing the DNA located in between, including the fljAB operon. It encompasses the genes blaTEM−1, strA-strB, sul2 and tet(B) responsible for the R-type ASSuT together with genes involved in plasmid replication and orfs of unknown function characteristically located on IncH1 plasmids. Its location and internal structure is fairly conserved in S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 strains regardless of the isolation source or country. Hence, in the S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 clonal lineage widespread in Germany, Switzerland and Italy, a resistance region derived from IncH1 plasmids has replaced the chromosomal region encoding the second flagellar phase and is an example of the stabilization of new plasmid-derived genetic material due to integration into the bacterial chromosome.

Emergence of NDM-1-producing Acinetobacter baumannii in China

To investigate the prevalence of bla(NDM-1) in Gram-negative bacilli in China. A total of 11 298 clinical Gram-negative bacilli, covering Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, were collected for PCR-based surveillance of bla(NDM-1) from 57 hospitals representing 18 provinces in China. For bla(NDM-1)-positive isolates, antibiotic susceptibilities were assessed and molecular typing was performed using PFGE. The genetic location of bla(NDM-1) was determined by analysis of PFGE profiles of S1 nuclease-digested genomic DNA and Southern blot hybridization. Plasmid transfer to E. coli recipients was investigated using filter mating and electroporation. Four A. baumannii isolates with bla(NDM-1) were identified in four different provinces in China: no positive isolates were detected among E. coli, K. pneumoniae and P. aeruginosa. These bla(NDM-1)-positive A. baumannii were resistant to all carbapenems and cephalosporins, and three remained susceptible to fluoroquinolones, aminoglycosides and colistin. The four NDM-1-producing A. baumannii were clonally diverse and carried bla(NDM-1) on different plasmids. Plasmids carrying bla(NDM-1) were successfully transferred from three of the four isolates to E. coli recipients, although the transconjugants and transformants were prone to lose the transferred plasmids after passage in the absence of ampicillin selection. We describe the emergence of A. baumannii producing NDM-1 in China. Systemic surveillance network should be established for monitoring these resistant bacteria.

IS6100-mediated genetic rearrangement within the complex class 1 integron In104 of the Salmonella genomic island 1

IS6100-mediated genetic rearrangement within the complex class 1 integron In104 of the Salmonella genomic island 1

RsLFY, a LEAFY homologue gene in radish (Raphanus sativus), is continuously expressed in vegetative, reproductive and seed development

The floral meristem identity gene LEAFY (LFY) in Arabidopsis plays a key role in flower development. We isolated two LFY-like genes from radish, designated RsLFY1 and RsLFY2. Comparison of genomic and cDNA sequences revealed that the number and positions of introns are precisely conserved in RsLFY1, RsLFY2 and LFY. Using RsLFY1 full- length cDNA as a probe, genomic DNA blot hybridization analysis detected two hybridizing fragments under high stringency, suggesting that there are two RsLFY loci within radish genome. Both genes were expressed during vegetative growth, reproductive growth, flower development and seed development. RsLFY1 was expressed slightly in leaf primordia and strongly in early floral meristem. It was expressed successively in primordia of sepals, petals, stamens, gynoecium, ovule integument and mature seeds. These results suggest that RsLFY1 plays a similar role to LFY in floral initiation and the developmental stages of floral organs, and that it also may regulate radish seed development, unlike LFY.

Identification of a tyrosinase (TYR) exon 4 deletion in albino ferrets (Mustela putorius furo)

Albinism is due to a lack of pigmentation in hair, skin and eye, and has been shown to occur in several animal species. Mutations of the tyrosinase (TYR) gene account for albinism in domestic cats, rabbits, cattle, mice and rats. In this study, we demonstrate that a TYR mutation accounts for albinism in the ferret (Mustela putorius furo). The coding sequence of the five exons of TYR was determined in genomic DNA from wild-type pigmented 'sable' coloured and albino ferrets. It was not possible to amplify TYR exon 4 in albino ferrets originating from different breeds. The deletion of exon 4 in albino ferrets was confirmed by Southern blot hybridization of genomic DNA from albino and pigmented ferrets. This is the first report of a deletion of a TYR exon in a non-human mammal.

Maize Pollen Coat Xylanase Facilitates Pollen Tube Penetration into Silk during Sexual Reproduction

Cell wall hydrolases are well documented to be present on pollen, but their roles on the stigma during sexual reproduction have not been previously demonstrated. We explored the function of the tapetum-synthesized xylanase, ZmXYN1, on maize (Zea mays L.) pollen. Transgenic lines (xyl-less) containing little or no xylanase in the pollen coat were generated with use of an antisense construct of the xylanase gene-coding region driven by the XYN1 gene promoter. Xyl-less and wild-type plants had similar vegetative growth. Electron microscopy revealed no appreciable morphological difference in anther cells and pollen between xyl-less lines and the wild type, whereas immunofluorescence microscopy and biochemical analyses indicated an absence of xylanase on xyl-less pollen. Xyl-less pollen germinated as efficiently as wild-type pollen in vitro in a liquid medium but less so on gel media of increasing solidity or on silk, which is indicative of partial impaired water uptake. Once germinated in vitro or on silk, the xyl-less and wild-type pollen tubes elongated at comparable rates. Tubes of germinated xyl-less pollen on silk did not penetrate into the silk as efficiently as tubes of wild-type pollen, and this lower efficiency could be overcome by the addition of xylanase to the silk. For wild-type pollen, coat xylanase activity on oat spelled xylan in vitro and tube penetration into silk were inhibited by xylose but not glucose. The overall findings indicate that maize pollen coat xylanase facilitates pollen tube penetration into silk via enzymatic xylan hydrolysis.

Molecular cloning and characterization of the rubber elongation factor gene and its promoter sequence from rubber tree (Hevea brasiliensis): A gene involved in rubber biosynthesis

Hevea rubber tree (Hevea brasiliensis) is the only plant species being cultivated for commercial production of rubber in the world. In order to meet ever increasing rubber demand, it is a prerequisite to identify and characterize a key gene involved in rubber biosynthesis and over-expression of rubber biosynthesis gene will eventually lead to enhance the latex (rubber) production in transgenic Hevea plants. Rubber elongation factor (REF) is a major protein located on the surface of large rubber particles in latex and is involved which is involved in rubber biosynthesis in H. brasiliensis. We report here cloning and characterization of REF gene as well as its 5′ promoter region from Hevea. REF gene (1367bp) has three exons interrupted by two introns and encoded a 138 amino acid peptide containing an open reading frame of 414bp with a calculated MW of 14,700Da. Nucleotide sequence analysis showed that 1.3kb genomic DNA showed 100% homology to REF cDNA from Hevea. Southern blot hybridization of genomic DNA with REF gene probe revealed that REF gene is encoded by a small gene family consisting of two members. RNA blot analysis indicated that REF transcript is highly expressed in high yielding clone than in low yielder. The cloned 5′ promoter region has a putative TATA element at −150 and CAAT box at −221 position. To identify the regulatory role of REF promoter, chimaeric fusion between REF promoter sequence and the β-glucuronidase (GUS) coding, uidA gene was constructed and used to transform tobacco and Arabidopsis. Expression of the uidA reporter gene was detected histochemically in the transformed tobacco plants where, GUS activity was detected in the leaf and petiole of transformed plants. The stable integration of REF:uidA fusion into the tobacco genome was further confirmed by PCR amplification and Southern blot analysis. A histochemical study of stable transformants demonstrated that the 5′ upstream region of REF can drive strong GUS gene expression specifically in the vascular tissues (xylem and phloem) of leaf, stem and midribs of transgenic Arabidopsis. GUS staining revealed that REF:GUS expression was also induced by wounding. The results suggested that the cloned REF promoter is capable of directing gene expression. Our ultimate goal is to produce transgenic Hevea plants with enhanced latex yield by over expression of REF protein.

Gene amplification of the Hps locus in Glycine max

BackgroundHydrophobic protein from soybean (HPS) is an 8 kD cysteine-rich polypeptide that causes asthma in persons allergic to soybean dust. HPS is synthesized in the pod endocarp and deposited on the seed surface during development. Past evidence suggests that the protein may mediate the adherence or dehiscence of endocarp tissues during maturation and affect the lustre, or glossiness of the seed surface.ResultsA comparison of soybean germplasm by genomic DNA blot hybridization shows that the copy number and structure of the Hps locus is polymorphic among soybean cultivars and related species. Changes in Hps gene copy number were also detected by comparative genomic DNA hybridization using cDNA microarrays. The Hps copy number polymorphisms co-segregated with seed lustre phenotype and HPS surface protein in a cross between dull- and shiny-seeded soybeans. In soybean cultivar Harosoy 63, a minimum of 27 ± 5 copies of the Hps gene were estimated to be present in each haploid genome. The isolation and analysis of genomic clones indicates that the core Hps locus is comprised of a tandem array of reiterated units, with each 8.6 kb unit containing a single HPS open reading frame.ConclusionThis study shows that polymorphisms at the Hps locus arise from changes in the gene copy number via gene amplification. We present a model whereby Hps copy number modulates protein expression levels and seed lustre, and we suggest that gene amplification may result from selection pressures imposed on crop plants.

Isolation of recombinant field strains of Marek’s disease virus integrated with reticuloendotheliosis virus genome fragments

Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.

A protocol for efficient biolistic transformation of mothbeanVigna aconitifolia L. Jacq. Marechal

A protocol for efficient direct gene transfer by using particle gun bombardment was developed for mothbeanVigna aconitifolia L. Jacq. Marechal. Hypocotyl explants from 2 cultivars of mothbean were transformed with 3 plasmids: pBI121, pHS101, and pHS102. Stable transformants were regenerated on MS medium supplemented with benzyladenine, α-naphthaleneacetic acid, and kanamycin. The helium pressure, plasmid type, and cultivar that were used determined the stable transformation frequency. Complete plants were regenerated and transferred to soil. The integration of the stable transgenes and reporter genes in plant genomes was shown by means of PCR amplification of these genes from plant genomic DNA and Southern blot hybridization with gene-specific probes. This method allows high-efficiency production of transgenic plants in mothbean.

Molecular Analysis of Fragile X Syndrome

The gene responsible for fragile X syndrome, fragile X mental retardation-1 (FMR1), contains an unstable repeat sequence of (CGG)(n). Additionally, an upstream promoter region of the gene--a CpG island--is abnormally methylated in most affected individuals. Amplification of CGG repeats and abnormal methylation show a correlation with affected status. The first basic and alternate protocols in this unit outline the use of the polymerase chain reaction (PCR) to rapidly detect the size of the repeat amplification. The second basic protocol describes the assessment of fragile X syndrome by direct Southern blot hybridization of genomic DNA digested with a pair of restriction enzymes, one of which is methylation-sensitive. Extent of amplification and level of methylation can be simultaneously detected. A combination of the two techniques can be used to characterize the genotypes of individual members in identified fragile X families.

A simple method for DNA extraction from marine bacteria that produce extracellular materials

We present a simple method for extracting DNA from the marine bacteria Hahella chejuensis, a Streptomyces sp., and a Cytophaga sp. Previously, DNA purification from these strains was hindered by the presence of extracellular materials. In our extraction method, the marine bacteria are lysed by freezing and grinding in liquid nitrogen, and treated with SDS. The extracted DNA is purified using a phenol/chloroform mixture, and precipitated in isopropanol. The extracted DNA is of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion, genomic DNA blot hybridization, and genomic DNA library construction. We used this method to extract genomic DNA from several other marine bacteria. Our method is a reproducible, simple, and rapid technique for routine DNA extractions from marine bacteria. Furthermore, the low cost of this method makes it attractive for large-scale studies.

Usefulness of double gene construct for rapid identification of transgenic mice exhibiting tissue‐specific gene expression

Identification of transgenics still requires PCR and genomic Southern blot hybridization of genomic DNA isolated from tail pieces. Furthermore, identification of transgene-expressing transgenics (hereafter called “expressor”) requires mRNA analyses (RT-PCR and Northern blot hybridization) or protein analysis (Western blotting and immunohistochemical staining using specific antibodies). These approaches are often labor-intensive and time-consuming. We developed a technique that simplifies the process of screening expressor transgenics using enhanced green fluorescent protein (EGFP), a noninvasive reporter recently utilized in a variety of organisms, including mice, as a tag. We constructed a MNCE transgene consisting of two expression units, MBP-NCre (termed “MN”) and CAG-EGFP (termed “CE”). MN consists of a myelin basic protein (MBP) promoter and NCre gene (Cre gene carrying a nuclear localization signal (NLS) sequence at its 5′ end). CE consists of a promoter element, CAG composed of cytomegalovirus (CMV) enhancer and chicken β-actin promoter, and EGFP cDNA. Of a total of 72 F0 mice obtained after pronuclear injection of MNCE at 1-cell egg stage, 15 were found to express EGFP when the tail, eye, and inner surface of the ear were inspected for EGFP fluorescence under UV illumination at weaning stage. These fluorescent mice were found to possess MNCE and to express NCre mRNA in a brain-specific manner. Mice exhibiting no fluorescence were transgenic or nontransgenic. Mice carrying MNCE, but exhibiting no fluorescence, never expressed NCre mRNA in any organs tested. These findings indicate that (i) direct inspection of the surface of mice for fluorescence under UV illumination enables identification of expressor transgenics without performances of the molecular biological analyses mentioned above, and (ii) systemic promoters such as CAG do not affect the tissue-specificity of a tissue-specific promoter such as MBP promoter, which is located upstream of CAG by approximately 2 kb. Mol. Reprod. Dev. 60: 446–456, 2001. © 2001 Wiley-Liss, Inc.

Usefulness of double gene construct for rapid identification of transgenic mice exhibiting tissue-specific gene expression.

Identification of transgenics still requires PCR and genomic Southern blot hybridization of genomic DNA isolated from tail pieces. Furthermore, identification of transgene-expressing transgenics (hereafter called "expressor") requires mRNA analyses (RT-PCR and Northern blot hybridization) or protein analysis (Western blotting and immunohistochemical staining using specific antibodies). These approaches are often labor-intensive and time-consuming. We developed a technique that simplifies the process of screening expressor transgenics using enhanced green fluorescent protein (EGFP), a noninvasive reporter recently utilized in a variety of organisms, including mice, as a tag. We constructed a MNCE transgene consisting of two expression units, MBP-NCre (termed "MN") and CAG-EGFP (termed "CE"). MN consists of a myelin basic protein (MBP) promoter and NCre gene (Cre gene carrying a nuclear localization signal (NLS) sequence at its 5' end). CE consists of a promoter element, CAG composed of cytomegalovirus (CMV) enhancer and chicken beta-actin promoter, and EGFP cDNA. Of a total of 72 F0 mice obtained after pronuclear injection of MNCE at 1-cell egg stage, 15 were found to express EGFP when the tail, eye, and inner surface of the ear were inspected for EGFP fluorescence under UV illumination at weaning stage. These fluorescent mice were found to possess MNCE and to express NCre mRNA in a brain-specific manner. Mice exhibiting no fluorescence were transgenic or nontransgenic. Mice carrying MNCE, but exhibiting no fluorescence, never expressed NCre mRNA in any organs tested. These findings indicate that (i) direct inspection of the surface of mice for fluorescence under UV illumination enables identification of expressor transgenics without performances of the molecular biological analyses mentioned above, and (ii) systemic promoters such as CAG do not affect the tissue-specificity of a tissue-specific promoter such as MBP promoter, which is located upstream of CAG by approximately 2 kb.

Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm

We report efficient germ-line transformation in the yellow fever mosquito Aedes aegypti accomplished using the piggyBac transposable element vector pBac[3xP3-EGFP afm]. Two transgenic lines were established and characterized; each contained the Vg-Defensin A transgene with strong eye-specific expression of the enhanced green fluorescent protein (EGFP) marker gene regulated by the artificial 3xP3 promoter. Southern blot hybridization and inverse PCR analyses of genomic DNA demonstrated a precise piggyBac-mediated, single copy insertion of the pBac[3xP3-EGFP afm, Vg-DefA] transposon in each transgenic line. For each line, genetic analysis confirmed stability and integrity of the entire transposon construct in the mosquito genome through the G 2–G 6 generations. Successful establishment of homozygous transgenic lines indicated that in both cases a non-lethal integration of the transposon into the mosquito genome had occurred. The 3xP3-EGFP marker was tested in mosquitoes with different genetic backgrounds. In white-eyed transgenic mosquitoes, the strong eye-specific expression of GFP was observed throughout all stages of development, starting from newly hatched first instar larvae to adults. A similar level and pattern of fluorescence was observed in red-eyed mosquitoes that were generated by crossing the 3xP3-EGFP transformants with the kh w white-eye mosquitoes transformed with the Drosophila cinnabar gene. Importantly, the utility of the 3xP3-EGFP, as marker gene for transformation of wild type mosquitoes, was demonstrated by strong eye-specific GFP expression in larval and pupal stages of black-eyed hybrids of the 3xP3-EGFP white-eye transformants and the wild type Rockefeller/UGAL strain. Finally, analysis of the Vg-DefA transgene expression in transformants from two established lines demonstrated strong blood-meal activation and fat-body-specific expression regulated by the Vg 1.8-kb 5′ upstream region.

Differential expression of the fibroin gene in developmental stages of silkworm, Antheraea mylitta (Saturniidae)

Fibroin gene expression during the larval developmental stages of the Saturniid silkworm, Antheraea mylitta, was analyzed. Northern blot analysis of larval silk gland total RNA using the fibroin gene as a probe showed that fibroin is expressed in the intermoult stages and repressed during the moulting stages. Abundance of fibroin transcripts gradually increased from the third to fifth intermoult stage, reaching a peak in the fifth intermoult. Transcripts declined during the early spinning stage. Western blot analysis of fibroin protein production with anti-fibroin antibody confirmed the differential fibroin expression, in accordance with fibroin mRNA synthesis. Dot blot hybridization of genomic DNA isolated from each larval developmental stage with the labelled fibroin gene showed that at the genomic level, the relative concentration of the fibroin gene was constant throughout the developmental stages. Our data confirm that fibroin gene expression in A. mylitta, like in B. mori, is transcriptionally controlled and shows differential temporal variations.

Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders.

Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA 105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66 bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration.

Cloning and Characterization of a Golgin-Related Gene from the Large-Scale Polymorphism Linked to the PML Gene

Megabase-scale mapping of the PML gene locus revealed the presence of a large-scale insertion–deletion polymorphism located 25 kb downstream of the PML gene. The polymorphism is organized as a head-to-tail tandem 25-kb repeat containing one to five units. Characterization of the first repeat unit downstream of PML revealed the presence of a gene with strong homology to a family of Golgin-related proteins. The gene, designated GLP (for Golgin linked to PML), is strongly expressed as a 6-kb transcript in normal human testis. In situ hybridization of normal human testis demonstrated that the expression of GLP was restricted to late meiotic germ cells. There was weak expression in late pachytene spermatocytes and strong expression in spermatids. GLP is 50% homologous to other Golgin-related proteins including the vesicle docking protein GM130. Southern blot hybridization of genomic DNA with a GLP probe demonstrated numerous homologous bands outside the PML locus. Three of these loci have been mapped by fluorescence in situ hybridization to chromosome loci 9q34.1, 15q11–q13, and 15q22–q24. Hybridization of a GLP cDNA probe to a zoo blot demonstrated multiple signals in nonhuman primates but not in other species and suggested the duplication of an ancestral locus around 20 million years ago.