Development of new methods in human gene mapping: selection for fragments of the human Y chromosome after chromosome-mediated gene transfer.

Chromosome-mediated gene transfer (CMGT) can be used to generate fragments of human chromosomes and chromosomal maps can be constructed using these fragments. In previous experiments CMGT techniques have been limited to those regions of the genome which encode biochemically selectable markers. We have extended the regions of the human genome which can be subjected to CMGT methods by employing a cell surface antigen as a selectable marker. These experiments have been facilitated by the discovery that co-transformation of chromosomes with a plasmid bearing a biochemically selectable marker followed by selection for the marker pre-selects for cells which have incorporated chromosomal fragments. The plasmid may also integrate into the donor chromosomes and this provides, in some cases, an additional selectable marker in the chromosome fragment of interest. Using these methods we have isolated for the first time cells containing varying portions of the human Y chromosome.     

Evolving strategies for making physical maps of mammalian chromosomes

Two types of physical maps are described: restriction maps made by top down approaches using enzymes that cut the genome infrequently, and complete libraries, made by bottom up approaches using fingerprinting of randomly selected cloned DNA. Construction of such maps for mammalian chromosomes is complicated by the mosaic nature of mammalian genomes, and extensive polymorphisms at the cleavage sites of most enzymes that yield large DNA fragments. However, it appears that both of these potential difficulties can be turned into advantages by new mapping strategies. When combined with yeast artificial chromosome cloning and polymerase chain reaction amplification methods, these approaches should soon yield complete maps of many human chromosomes.     

PFGE-resolved RFLP analysis and long range restriction mapping of the DNA of Arabidopsis thaliana using whole YAC clones as probes.

The cleavage patterns of 23 rare-cutting restriction endonucleases (rcREs) on high molecular weight DNA, isolated from leaves of Arabidopsis thaliana (Arabidopsis), have been analysed using pulsed field gel electrophoresis (PFGE). The DNA digested with rcREs can be used for restriction fragment length polymorphism (RFLP) analysis. We show that RFLPs are more readily identified in restriction fragments that require resolution by PFGE than in smaller restriction fragments. Taking advantage of the low dispersed repetitive DNA content of the Arabidopsis genome, whole yeast artificial chromosomes (YACs) were used as probes to PFGE resolved genomic DNA. This enabled whole YAC clones to be used as RFLP markers and long range restriction maps to be constructed. These techniques should enhance the analysis of regions of the genome of Arabidopsis (and other organisms with low levels of dispersed repetitive DNA) that are the subject of chromosome walking strategies to isolate particular loci.     

A NheI macrorestriction map of the Neisseria meningitidis B1940 genome

Abstract A macrorestriction map of the Neisseria meningitidis strain B1940 genome was constructed by two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. Digestion of the genomic DNA with the restriction endonuclease NHe I revealed 15 fragments between 10 kb and 450 kb. The sum of the fragments and resolution of the linearized chromosome yielded a total genome size of about 2.3 Mbp. By overlapping methylation with the Alu I-methylase six Nhe I recognition sites could be blocked. Fragments were ordered by partial/complete 2D-PFGE of genomic DNA with and without prior Alu I methylation, respectively. All nine Alu I-methylase/ Nhe I and 14 Nhe I restriction sites could be mapped on a single circular chromosome. This map will serve as a useful tool for further genetic analysis of meningococci and exemplifies the power of non-radioactive 2D-PFGE techniques to construct large physical genome maps with a single restriction enzyme.     

Fine mapping of inherent flexibility variation along DNA molecules. Validation by atomic force microscopy (AFM) in buffer

Curvature and flexibility are structural properties of central importance to genome function. However, due to the difficulties in finding suitable experimental conditions, methods for studying one without the interference of the other have proven to be difficult. We propose a new approach that provides a measure of inherent flexibility of DNA by taking advantage of two powerful techniques, X-ray crystallography and nuclear magnetic resonance. Both techniques are able to detect local curvature on DNA fragments but, while the first analyzes DNA in the solid state, the second works on DNA in solution. Comparison of the two data sets allowed us to calculate the relative contribution to flexibility of the three rotations and three translations, which relate successive base pair planes for the ten different dinucleotide steps. These values were then used to compute the variation of flexibility along a given nucleotide sequence. This allowed us to validate the method experimentally through comparisons with maps of local fluctuations in DNA molecule trajectory constructed from atomic force microscopy imaging in solution. We conclude that the six dinucleotide-step parameters defined here provide a powerful tool for the exploration of DNA structure and, consequently will make an important contribution to our understanding of DNA-sequence-dependent biological processes.     

Genome mapping by arbitrary amplification of yeast artificial chromosomes

Several methods have been described for using the polymerase chain reaction (PCR) to isolate fragments of DNA for genome mapping. We have developed an approach for isolating discrete fragments by amplifying DNA with single oligonucleotides (10-mers) with arbitrarity selected sequences. The method is rapid and technically simple. We isolated fragments from a contig of three yeast artificial chromosomes (YACs) from the human Xq28 chromosomal region. We purified YACs yWXD 37, yWXD348, and yWXD705 from a preparative pulsed field gel. Amplifications of each YAC were performed with single 10-mers as the PCR primers and the products were visualized on agarose gels. These fragments have been successfully used as hybridization probes against Southern blots containing the YACs and against blots containing human genomic DNA and somatic cell hybrids containing Xq28 as their only human constituent. The results have been concordant with the known order of the YACs. We have also successfully combined 10-mers with primers derived from vector arm sequences to isolate YAC ends. We discuss several uses of this method in comparative mapping and in filling in gaps in physical and genetic maps.     

Cleavage maps for human cytomegalovirus DNA strain AD169 for restriction endonucleases EcoRI, BglII, and HindIII.

We have used cloned EcoRI fragments of the human CMV (HCMV) genome, strain AD169, to prepare restriction endonuclease maps of the DNA. Individual 32P-labeled cloned fragments were hybridized to Southern blots of HCMV DNA cleaved to completion with the restriction endonucleases BglII and HindIII and cleaved partially with EcoRI. By determining which EcoRI fragments hybridized to the same band on a Southern blot, we were able to establish linkage groups. This information coupled with the data derived from digestion of the cloned fragments with the enzymes BglII and HindIII (Tamashiro et al., J. Virol. 42:547-557, 1982) provided the basis for the construction of detailed maps for the enzymes EcoRI, BglII, and HindIII. We also identified the EcoRI fragments derived from the termini of this genome and mapped them with respect to the BglII and HindIII terminal fragments. From our mapping data, we conclude that the genome of HCMV is approximately 240 kilobases in length and is divided into long (198 kilobases) and short (42 kilobases) regions. Both regions consist of a unique sequence bounded by inverted repeats (11 to 12 kilobases for the long region and 2 to 3 kilobases for the short region). Furthermore, the long and short regions can invert relative to each other.     

Human cytomegalovirus DNA: BamHI, EcoRI and PstI restriction endonuclease cleavage maps

The cloned HindIII fragments of human cytomegalovirus (HCMV) strain AD169 DNA were mapped with respect to the BamHI, EcoRI and PstI restriction endonuclease cleavage sites. Composite restriction endonuclease cleavage maps for the entire virus genome were constructed using the previously established linkages between the HindIII fragments.     

Identifying co‐opted transposable elements using comparative epigenomics

The human genome gives rise to different epigenomic landscapes that define each cell type and can be deregulated in disease. Recent efforts by ENCODE, the NIH Roadmap and the International Human Epigenome Consortium (IHEC) have made significant advances towards assembling reference epigenomic maps of various tissues. Notably, these projects have found that approximately 80% of human DNA was biochemically active in at least one epigenomic assay while only approximately 10% of the sequence displayed signs of purifying selection. Given that transposable elements (TEs) make up at least 50% of the human genome and can be actively transcribed or act as regulatory elements either for their own purposes or be co‐opted for the benefit of their host; we are interested in exploring their overall contribution to the “functional” genome. Traditional methods used to identify functional DNA have relied on comparative genomics, conservation analysis and low throughput validation assays. To discover co‐opted TEs, and distinguish them from noisy genomic elements, we argue that comparative epigenomic methods will also be important.     

8. Molecular biology and genetic conservation programmes

The techniques of molecular biology will become a standard part of germplasm conservation and exploitation. They are being used to gather information very rapidly about chromosome structure and genetic variation within the major crop species. Genetic maps with hundreds of DNA sequence markers covering the whole genome have already been created for some crops, such as maize, soybean, wheat and potato. Genetic variation is being revealed by the combined use of restriction endonucleases, fractionation of DNA fragments by electrophoresis and investigation of the size of specific allelic fragments. This kind of approach offers new opportunities to assess the extent of genetic variation among accessions in germplasm collections, thereby helping to decide which accessions are essentially duplicates and which should be maintained in a core collection. I recommend that germplasm banks will in the future also contain diagnostic DNA markers for characterizing and screening germplasm.
When material from germplasm banks is used in crop plant breeding programmes to transfer specific traits into the crop, the availability of a complete set of molecular markers covering the entire genome makes it straightforward to discover which segments have been transferred and which are essential to maintain, so as to preserve the introduced trait.
Germplasm banks are obviously a source of new genetic variation for the molecular geneticist as well as the plant breeder. The isolation of specific alleles determining self-incompatibility from Brassica oleracea accessions for subsequent introduction into oil seed rape is described as an example.     

Codominant PCR-based markers for Pinus taeda developed from mapped cDNA clones

 We report a strategy for developing codominant PCR-based genetic markers by using sequenced cDNA clones from loblolly pine (Pinus taeda L.). These clones were previously used as probes for detecting restriction fragment length polymorphisms (RFLPs) to generate linkage maps. After assessing the complexity of banding patterns from Southern blots, we selected clones representing relatively simple gene families, and then determined nucleotide sequences for about 200 bp at each end of the cDNA inserts. Specific PCR primers were designed to amplify samples of genomic DNA derived from two loblolly pine mapping populations. Polymorphisms were detected after digesting the amplified DNA fragments with a battery of restriction endonucleases, and most polymorphisms were inherited in a Mendelian fashion. These newly identified genetic markers are codominant and relatively simple to use. By assaying DNA from individuals used to construct RFLP maps, we show that most of these markers map to the same position as the RFLP loci detected using their corresponding cDNAs as probes, implying that these markers have been converted from RFLP to PCR-based methods. These PCR-based markers will be useful for genome mapping and population genetics. Received: 10 February 1998 / Accepted: 25 February 1998     

Current status and future possibilities of molecular genetics techniques in <Emphasis Type="Italic">Brassica napus</Emphasis>

As PCR methods have improved over the last 15 years, there has been an upsurge in the number of new DNA marker tools, which has allowed the generation of high-density molecular maps for all the key Brassica crop types. Biotechnology and molecular plant breeding have emerged as a significant tool for molecular understanding that led to a significant crop improvement in the Brassica napus species. Brassica napus possess a very complicated polyploidy-based genomics. The quantitative trait locus (QTL) is not sufficient to develop effective markers for trait introgression. In the coming years, the molecular marker techniques will be more effective to determine the whole genome impairing desired traits. Available genetic markers using the single-nucleotide sequence (SNP) technique and high-throughput sequencing are effective in determining the maps and genome polymorphisms amongst candidate genes and allele interactions. High-throughput sequencing and gene mapping techniques are involved in discovering new alleles and gene pairs, serving as a bridge between the gene map and genome evaluation. The decreasing cost for DNA sequencing will help in discovering full genome sequences with less resources and time. This review describes (1) the current use of integrated approaches, such as molecular marker technologies, to determine genome arrangements and interspecific outcomes combined with cost-effective genomes to increase the efficiency in prognostic breeding efforts. (2) It also focused on functional genomics, proteomics and field-based breeding practices to achieve insight into the genetics underlying both simple and complex traits in canola.     

Buccal cells as a source of DNA for comparative animal genomic analysis

The source of DNA of adequate quality and quantity is an important consideration in genome analysis. In many animal and livestock species, easy access to DNA will facilitate the rapid and reliable genotyping of a large number of individual individuals. Here, we describe the use, for the first time, of buccal cells from non-human mammalian species as a source of DNA template for PCR and restriction analysis. The buccal cells from the pig, cow and human, were used to amplify PCR fragments that were scanned SNPs and for comparative genome analysis. The work indicates that buccal cells are also adequate sources of DNA for genome analysis of animals that have been identified as priorities in comparative genomics.     

Applying a new generation of genetic maps to understand human inflammatory disease

The sequencing of the human genome and the intense study of its variation in different human populations have improved our understanding of the genome's architecture. It is now becoming clear that segments of the genome that are unbroken by reshuffling or recombination during meiosis create a mosaic of DNA 'haplotype blocks'. Here, we discuss the advantages and limitations of this block structure. Haplotype blocks hold the promise of reducing the complexity of analysing the human genome for association with disease. But can they deliver on this promise? First generation maps of these block patterns, such as the admixture and haplotype maps, are now emerging and, it is to be hoped, will accelerate the discovery of alleles that contribute to susceptibility to human inflammatory diseases.     

Direct sequencing of RAPD fragments using 3'-extended oligonucleotide primers and dye terminator cycle-sequencing.

Random amplified polymorphic DNA (RAPD) markers are used widely to develop high resolution genetic maps and for genome fingerprinting. Typically, single oligomers of approximately 10 nucleotides are used to PCR amplify characteristic RAPD marker fragments. We describe an efficient method for the direct end-sequencing of gel-purified RAPD fragments using one primer from a set of four 3'-terminal extended (A, T, C or G) oligonucleotides, identical to the RAPD primer but for the single nucleotide extension. Strand-specific DNA sequence could be independently read from each of the RAPD fragments without recourse to strand separation or fragment cloning. Informative RAPD fragments could be readily converted into mapped STS or SCAR loci using this technology. The 3'-extended primers may also be used to amplify independent genomic RAPD markers.     

Molecular cloning and physical mapping of murine cytomegalovirus DNA.

Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.     

Physical maps for Herpes simplex virus type 1 DNA for restriction endonucleases Hind III, Hpa-1, and X. bad.

It has been proposed that the genome of herpes simplex virus type 1 (HSV-1) consists of two internal unique sequences, S and L, bounded by two sets of redundant sequences (P. Sheldrick and N. Berthelot, 1974). In this arrangement, terminal sequences (TRs and TRl) are repeated in an internal inverted form (IRs and IRl) and delimit S and L. Furthermore, a body of evidence has accumulated that suggests that S and L themselves are inverted, giving rise to four related forms of the HSV genome. In this study the ordering of restruction endonuclease fragments of HSV-1 DNA for physical maps has been studied using molecular hybridization techniques and the cleavage of isolated restriction endonuclease fragments with further restriction endonucleases. Physical maps for the fragments produced by Hind III, Hpa-1, and X. bad have been constructed for the four related forms of the HSV-1 genome. TRs and IRs were found to be between 3.5 x 10(6) and 4.5 x 10(6) daltons, TRl and IRl about 6 x 10(6) daltons, S about 8 x 10(6) to 9 x 10(6) daltons, and L about 6.8 x 10(6) daltons.     

Molecular Cloning and Physical Mapping of Restriction Endonuclease Fragments of Autographa californica Nuclear Polyhedrosis Virus DNA

A restriction fragment library containing Autographa californica nuclear polyhedrosis virus (AcNPV) DNA was constructed by using the pBR322 plasmid as a vector. The library, which is representative of more than 95% of the viral genome, consists of 2 of the 7 BamHI fragments, 12 of the 24 HindIII fragments, and 23 of the 24 EcoRI fragments. The cloned fragments were characterized and used to generate physical maps of the genome by hybridizing nick-translated recombinant plasmid to Southern blots of AcNPV DNA digested with SmaI, BamHI, XhoI, PstI, HindIII, and EcoRI restriction endonucleases. This information was used to define our strain of AcNPV (HR3) with respect to other strains for which physical maps have been previously published. The hybridization data also indicate that reiteration of DNA sequences occurs at the HindIII-L and -Q regions of the genome.