Showing papers on "genomic DNA published in 1998"
TL;DR: The implementation of array CGH is demonstrated to be able to measure copy number with high precision in the human genome, and to analyse clinical specimens by obtaining new information on chromosome 20 aberrations in breast cancer.
Abstract: Gene dosage variations occur in many diseases. In cancer, deletions and copy number increases contribute to alterations in the expression of tumour-suppressor genes and oncogenes, respectively. Developmental abnormalities, such as Down, Prader Willi, Angelman and Cri du Chat syndromes, result from gain or loss of one copy of a chromosome or chromosomal region. Thus, detection and mapping of copy number abnormalities provide an approach for associating aberrations with disease phenotype and for localizing critical genes. Comparative genomic hybridization3(CGH) was developed for genome-wide analysis of DNA sequence copy number in a single experiment. In CGH, differentially labelled total genomic DNA from a 'test' and a 'reference' cell population are cohybridized to normal metaphase chromosomes, using blocking DNA to suppress signals from repetitive sequences. The resulting ratio of the fluorescence intensities at a location on the 'cytogenetic map', provided by the chromosomes, is approximately proportional to the ratio of the copy numbers of the corresponding DNA sequences in the test and reference genomes. CGH has been broadly applied to human and mouse malignancies. The use of metaphase chromosomes, however, limits detection of events involving small regions (of less than 20 Mb) of the genome, resolution of closely spaced aberrations and linking ratio changes to genomic/genetic markers. Therefore, more laborious locus-by-locus techniques have been required for higher resolution studies2,3,4,5. Hybridization to an array of mapped sequences instead of metaphase chromosomes could overcome the limitations of conventional CGH (ref. 6) if adequate performance could be achieved. Copy number would be related to the test/reference fluorescence ratio on the array targets, and genomic resolution could be determined by the map distance between the targets, or by the length of the cloned DNA segments. We describe here our implementation of array CGH. We demonstrate its ability to measure copy number with high precision in the human genome, and to analyse clinical specimens by obtaining new information on chromosome 20 aberrations in breast cancer.
2,317 citations
TL;DR: The technology and applications of microarrays of immobilized DNA or oligonucleotides are reviewed, which have been successfully applied to the simultaneous expression of many thousands of genes and to large-scale gene discovery, as well as polymorphism screening and mapping of genomic DNA clones.
Abstract: The technology and applications of microarrays of immobilized DNA or oligonucleotides are reviewed. DNA arrays are fabricated by high-speed robotics on glass or nylon substrates, for which labeled probes are used to determine complementary binding allowing massively parallel gene expression and gene discovery studies. Oligonucleotide microarrays are fabricated either by in situ light-directed combinational synthesis or by conventional synthesis followed by immobilization on glass substrates. Sample DNA is amplified by the polymerase chain reaction (PCR), and a fluorescent label is inserted and hybridized to the microarray. This technology has been successfully applied to the simultaneous expression of many thousands of genes and to large-scale gene discovery, as well as polymorphism screening and mapping of genomic DNA clones.
925 citations
TL;DR: A freely available computer program solves the problem of efficiently aligning a transcribed and spliced DNA sequence with a genomic sequence containing that gene, allowing for introns in the genomic sequence and a relatively small number of sequencing errors.
Abstract: We address the problem of efficiently aligning a transcribed and spliced DNA sequence with a genomic sequence containing that gene, allowing for introns in the genomic sequence and a relatively small number of sequencing errors. A freely available computer program, described herein, solves the problem for a 100-kb genomic sequence in a few seconds on a workstation.
764 citations
TL;DR: The attenuated phenotype of MVA is the result of numerous mutations, particularly affecting the host interactive proteins, including the ankyrin-like genes, but also involving some structural proteins.
581 citations
TL;DR: The accuracy of computational gene-finding methods has improved significantly, to the point where a reasonable approximation of the gene structures within an extended genomic region can often be predicted in advance of more detailed experimental studies.
552 citations
TL;DR: It is shown that human exons with flanking genomic DNA sequences can be classified into 12 mutually exclusive categories, which could serve as a standard for future studies so that direct comparisons of results can be made.
Abstract: To facilitate gene finding and for the investigation of human molecular genetics on a genome scale, we present a comprehensive survey on various statistical features of human exons. We first show that human exons with flanking genomic DNA sequences can be classified into 12 mutually exclusive categories. This classification could serve as a standard for future studies so that direct comparisons of results can be made. A database for eight categories (related to human genes in which coding regions are split by introns) was built from GenBank release 87.0 and analyzed by a number of methods to characterize statistical features of these sequences that may serve as controls or regulatory signals for gene expression. The statistical information compiled includes profiles of signals for transcription, splicing and translation, various compositional statistics and size distributions. Further analyses reveal novel correlations and constraints among different splicing features across an internal exon that are consistent with the Exon Definition model. This information is fundamental for a quantitative view of human gene organization, and should be invaluable for individual scientists to design human molecular genetics experiments.
444 citations
TL;DR: It is demonstrated that allelic variation in any two isolates of a species can be scanned, mapped, and scored directly and efficiently without allele-specific polymerase chain reaction, without creating new strains or constructs, and without knowing the specific nature of the variation.
Abstract: As more genomes are sequenced, the identification and characterization of the causes of heritable variation within a species will be increasingly important. It is demonstrated that allelic variation in any two isolates of a species can be scanned, mapped, and scored directly and efficiently without allele-specific polymerase chain reaction, without creating new strains or constructs, and without knowing the specific nature of the variation. A total of 3714 biallelic markers, spaced about every 3.5 kilobases, were identified by analyzing the patterns obtained when total genomic DNA from two different strains of yeast was hybridized to high-density oligonucleotide arrays. The markers were then used to simultaneously map a multidrug-resistance locus and four other loci with high resolution (11 to 64 kilobases).
397 citations
TL;DR: It is suggested that six pRNAs form a hexameric ring by the intermolecular interaction of two RNA loops to serve as part of the DNA transportation machinery.
335 citations
TL;DR: This chapter discusses DNA and RNA cleavage by metal complexes and the synthesis of compounds that cleave nucleic acids should help in the design of potential therapeutic agents for the treatment of cancer and viral diseases.
Abstract: Publisher Summary This chapter discusses DNA and RNA cleavage by metal complexes. DNA and RNA cleavage, a very active field of research, has been developed in two main and complementary directions within the past decade: oxidative cleavage and hydrolysis. In general, the difference between the two different approaches are (1) the preparation of new chemical tools to study genomic DNA. The recognition sites of most of the restriction enzymes are often limited to palindromic sequences, and it is useful to have artificial nucleases able to cleave DNA at any desired sequence, and (2), the synthesis of compounds that cleave nucleic acids should help in the design of potential therapeutic agents for the treatment of cancer and viral diseases. The challenging development of new, efficient DNA and RNA cleavage agents can require a strong cooperation between chemists, biochemists, and molecular biologists.
327 citations
TL;DR: The molecular cloning, tissue distribution, and chromosomal localization of novel genes encoding G-protein-coupled receptors (GPCRs) are reported, and the gene encoding GPR30 was localized to chromosome 7p22 and GPR35 to chromosome 2q37.
326 citations
TL;DR: The isolation procedure described here is relatively brief and relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent SDS.
Abstract: There are a number of different procedures for the preparation of genomic DNA. They all start with some form of cell lysis, followed by deproteination and recovery of DNA. The main differences between various approaches lie in the extent of deproteination and in molecular weight of the DNA produced. The isolation procedure described here is relatively brief and relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent SDS.
TL;DR: Evidence was obtained that changes were also brought about by DNA methylation in newly synthesized amphiploid plants, leading to genetic diploidization and gene-dosage compensation and thus increasing variation among individuals.
Abstract: We recently reported that formation of allopolyploid wheat was accompanied by rapid nonrandom changes in low-copy noncoding DNA sequences. In this report we show that following allopolyploidization, changes also occurred in coding sequences. Genomic DNA of nine different newly synthesized amphiploids of different ploidy levels and their parental lines was digested with five restriction enzymes and probed with 43 coding sequences. The sequences, 19 genomic and 24 cDNA sequences, are group (homoeologous) specific and represent the proximal and distal regions of the short and long arms of the seven homoeologous groups of the Triticeae. We revealed three types of changes: disappearance of a parental hybridization fragment(s), appearance of a novel fragment(s), and simultaneous disappearance of a parental fragment(s) and appearance of a novel fragment(s). No elimination of sequences took place, since in every sequence studied the parental hybridization fragments were present in at least one of the enzyme digests. Variations in pattern among individual plants of the same amphiploid, as well as between several synthetic and natural amphiploids, indicated that at least some of the genomic changes occurred at random. Intergenomic recombination was not the cause of the observed changes. Evidence was obtained, however, that changes were also brought about by DNA methylation. Methylation may cause inactivation of genes or modify their expression levels in some of the newly synthesized amphiploid plants, leading to genetic diploidization and gene-dosage compensation and thus increasing variation among individuals.
TL;DR: Phylogenetic analysis of 16S ribosomal DNA (rDNA) clones obtained by PCR from uncultured bacteria inhabiting a wide range of environments has increased the knowledge of bacterial diversity, but the rRNA sequences detected possibly are common contaminants in reagents used to prepare genomic DNA.
Abstract: Phylogenetic analysis of 16S ribosomal DNA (rDNA) clones obtained by PCR from uncultured bacteria inhabiting a wide range of environments has increased our knowledge of bacterial diversity. One possible problem in the assessment of bacterial diversity based on sequence information is that PCR is exquisitely sensitive to contaminating 16S rDNA. This raises the possibility that some putative environmental rRNA sequences in fact correspond to contaminant sequences. To document potential contaminants, we cloned and sequenced PCR-amplified 16S rDNA fragments obtained at low levels in the absence of added template DNA. 16S rDNA sequences closely related to the genera Duganella (formerly Zoogloea), Acinetobacter, Stenotrophomonas, Escherichia, Leptothrix, and Herbaspirillum were identified in contaminant libraries and in clone libraries from diverse, generally low-biomass habitats. The rRNA sequences detected possibly are common contaminants in reagents used to prepare genomic DNA. Consequently, their detection in processed environmental samples may not reflect environmentally relevant organisms.
TL;DR: The AFLP technique has been extensively used with plant DNA for the development of high-resolution genetic maps and for the positional cloning of genes of interest, but its application is rapidly expanding in bacteria and higher eukaryotes for determining genetic relationships and for epidemiological typing.
Abstract: Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to DNAs of any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter and restriction site sequence, with additional nucleotides at the 3′-end, are used as selective agents to amplify a subset of ligated fragments. Polymorphisms are identified by the presence or absence of DNA fragments following analysis on polyacrylamide gels. This technique has been extensively used with plant DNA for the development of high-resolution genetic maps and for the positional cloning of genes of interest. However, its application is rapidly expanding in bacteria and higher eukaryotes for determining genetic relationships and for epidemiological typing. This review describes the AFLP procedure, and recent, novel applications in the molecular fingerprinting of DNA from both eukaryotic and prokaryotic organisms.
TL;DR: Data suggest that some of the RGA clones may hybridize to resistance genes, and some RFLPs were shown to map to genomic regions containing virus and fungus resistance genes.
Abstract: Many of the plant disease resistance genes that have been isolated encode proteins with a putative nucleotide binding site and leucine-rich repeats (NBS-LRR resistance genes). Oligonucleotide primers based on conserved motifs in and around the NBS of known NBS-LRR resistance proteins were used to amplify sequences from maize genomic DNA by polymerase chain reaction (PCR). Eleven classes of non-cross-hybridizing sequences were obtained that had predicted products with high levels of amino acid identity to NBS-LRR resistance proteins. These maize resistance gene analogs (RGAs) and one RGA clone obtained previously from wheat were used as probes to map 20 restriction fragment length polymorphism (RFLP) loci in maize. Some RFLPs were shown to map to genomic regions containing virus and fungus resistance genes. Perfect cosegregation was observed between RGA loci and the rust resistance loci rp1 and rp3. The RGA probe associated with rp1 also detected deletion events in several rp1 mutants. These data strongly suggest that some of the RGA clones may hybridize to resistance genes.
TL;DR: The cloning and characterization of a second member of the CYP19 family in goldfish indicates that a single locus encodes the brain-derived P450aromB (CYPl9B), whereas one or two different loci encode the ovarian form (CyPl9A).
Abstract: Evidence to date indicates that the gene encoding cytochrome P450 aromatase (P450arom) in humans is a single member of the CYPl9 family, but multiple CYPl9 loci and isoforms have been identified in pigs. Here we report the cloning and characterization of a second member of the CYP19 family in goldfish. A search for P450arom variants was prompted by studies showing that a full-length P450arom complementary DNA (cDNA) isolated from a goldfish brain cDNA library hybridizes with a high abundance 3 kb transcript in brain RNA but fails to detect a message in ovarian RNA. A stepwise PCR cloning strategy led to isolation of a 1.9-kb cDNA, which encodes a protein of 518 amino acids and has a predicted mol wt of 58.7K. The ovary-derived P450arom (-A) shares 68-72% sequence identity with ovarian aromatases of other fish species, but only 62% identity with the homologous brain-derived P450arom (-B). Amino acid differences are distributed throughout the two goldfish P450arom forms, but presumptive functional domains are highly conserved. Both P450aromA and -B are able to aromatize [3H]androgen to [3H]estrogen when expressed in nonsteroidogenic COS cells. Southern analysis and PCR-restriction analysis of genomic DNA using discriminating probes and primers indicates that a single locus encodes the brain-derived P450aromB (CYPl9B), whereas one or two different loci encode the ovarian form (CYPl9A). Northern blot analysis revealed two P450aromA messenger RNAs (1.9 >> 3.0 kb) in ovary. Simultaneous PCR amplification with A- and B-specific primer pairs confirms that P450aromA is the only form expressed in ovaries, but shows overlapping expression of the two genes in neural tissues. Whereas P450aromB messenger RNA predominates in brain (B/A, approximately 14:1), the ratios are reversed in retina (B/A, approximately 1:25). Further studies are required to resolve the evolutionary and functional implications of multiple CYPl9 genes and P450arom isozymes in goldfish, their differential expression in brain and ovary, and whether observations can be generalized to other vertebrates.
TL;DR: The use of PCR with degenerate oligonucleotide primers is an efficient method to identify numerous RGCs in plants and demonstrates the existence of clustered multigene families for each of the four RGC sequences.
Abstract: The recent cloning of genes for resistance against diverse pathogens from a variety of plants has revealed that many share conserved sequence motifs. This provides the possibility of isolating numerous additional resistance genes by polymerase chain reaction (PCR) with degenerate oligonucleotide primers. We amplified resistance gene candidates (RGCs) from lettuce with multiple combinations of primers with low degeneracy designed from motifs in the nucleotide binding sites (NBSs) of RPS2 of Arabidopsis thaliana and N of tobacco. Genomic DNA, cDNA, and bacterial artificial chromosome (BAC) clones were successfully used as templates. Four families of sequences were identified that had the same similarity to each other as to resistance genes from other species. The relationship of the amplified products to resistance genes was evaluated by several sequence and genetic criteria. The amplified products contained open reading frames with additional sequences characteristic of NBSs. Hybridization of RGCs to genom...
TL;DR: The isolation of two genes belonging to the RAD51 and DMC1 families from Arabidopsis thaliana indicated that not only AtRAD51 but also AtDMC1 are regulated during the cell cycle, with S-phase-specific induction.
Abstract: By using RT-PCR and degenerate oligonucleotides based on the sequence homology between the yeast RAD51 and DMC1 genes, two genes belonging to the RAD51 and DMC1 families were isolated from Arabidopsis thaliana ecotype Columbia. A RAD51 genomic DNA was also sequenced which is almost identical to its Landsberg erecta counterpart, except for a few translationally silent substitutions and for the presence of a 527-bp element downstream of the polyadenylation site. This element is repeated in the genome of Arabidopsis. Northern analyses were conducted to characterize the expression pattern of both these genes. AtRAD51 and AtDMC1 are expressed in flower buds, but also in the mitotically active cells from a suspension culture. AtRAD51, but not AtDMC1, transcript level increases after gamma irradiation of the cells. Finally, a synchronisation experiment conducted with the suspension culture indicated that not only AtRAD51 but also AtDMC1 are regulated during the cell cycle, with S-phase-specific induction. Since DMC1 genes have always been regarded as being specifically meiotic, we discuss the significance of this mitotic transcriptional regulation in Arabidopsis.
TL;DR: This technique provides a new way to develop molecular markers for assessing the genetic diversity of germplasm based upon potential candidate resistance genes in diverse species.
Abstract: Genes cloned from diverse plants for resistance to different pathogens have sequence similarities in domains presumably involved in pathogen recognition and signal transduction in triggering the defense response. Primers based on the conserved regions of resistance genes often amplify multiple fragments that may not be separable in an agarose gel. We used denaturing polyacrylamide-gel electrophoresis to detect PCR products of plant genomic DNA amplified with primers based on conserved regions of resistance genes. Depending upon the primer pairs used, 30–130 bands were detected in wheat, rice, and barley. As high as 47%, 40%, and 27% of the polymorphic bands were detected in rice, barley, and wheat, respectively, and as high as 12.5% of the polymorphic bands were detected by certain primers in progeny from a cross of the wheat cultivars ‘Stephens’ and ‘Michigan Amber’. Using F6 recombinant inbred lines from the ‘Stephens’בMichigan Amber’ cross, we demonstrated that polymorphic bands amplified with primers based on leucine-rich repeats, nucleotide-binding sites and protein kinase genes, were inherited as single loci. Linkages between molecular markers and stripe rust resistance genes were detected. This technique provides a new way to develop molecular markers for assessing the genetic diversity of germplasm based upon potential candidate resistance genes in diverse species.
TL;DR: Real-time 5' nuclease PCR assays to rapidly distinguish single-base polymorphism using a battery-powered miniature analytical thermal cycling instrument (MATCI) to demonstrate the feasibility of using the MATCI for diagnosis of infectious diseases and genetic disorders.
Abstract: This report describes real-time 5' nuclease PCR assays to rapidly distinguish single-base polymorphism using a battery-powered miniature analytical thermal cycling instrument (MATCI). Orthopoxviruses and the human complement component C6 gene served as targets to demonstrate the feasibility of using the MATCI for diagnosis of infectious diseases and genetic disorders. In the Orthopoxvirus assay, consensus Orthopoxvirus PCR primers were designed to amplify 266-281 base-pair (bp) segments of the hemagglutinin (HA) gene in camelpox, cowpox, monkeypox, and vaccinia viruses. A vaccinia virus-specific fluorogenic (TaqMan) probe was designed to detect a single-base (A/G) substitution within the HA gene. In the C6 gene assay, a 73-bp segment of the C6 gene was PCR-amplified from human genomic DNA, and TaqMan probes were used to detect a single-base (A/C) polymorphism in the second position of codon 98. The MATCI correctly identified the nucleotide differences in both viral DNA and human genomic DNA. In addition, using a rapid DNA preparation method, it was possible to achieve sample, preparation of human genomic DNA, DNA amplification, and real-time detection in less than 1 h.
Patent•
27 Nov 1998TL;DR: In this article, the base cytosine (not 5-methyl-cytosine) in a genomic DNA taken from any tissue sample is converted into uracil through treatment with a bisulphite solution.
Abstract: The invention relates to a method for characterising, classifying and distinguishing tissues and cell types, for predicting the behaviour of tissues and groups of cells, and for identifying genes which have altered in their expression. Said method is characterised in that the base cytosine (not 5-methyl-cytosine) in a genomic DNA taken from any tissue sample is converted into uracil through treatment with a bisulphite solution. Fractions of the genomic DNA which has been treated are amplified by using very short or degenerated oligonucleotides, and the remaining cytosines of the amplified fractions are detected by means of hybridisation or polymerase reaction. The data generated from the analysis and automatically transferred to a processing algorithm is then used to draw conclusions as to the phenotype of the cell material which was analysed.
Journal Article•
TL;DR: The results suggest that this mouthwash procedure may be suitable for large community-based studies of genetic susceptibility to disease in which samples can be collected by the participants themselves, mailed back to the study center, and stored for months prior to DNA analysis.
Abstract: Genomic DNA for genetic analyses has traditionally been derived from blood samples. With the availability of PCR techniques requiring only minute amounts of DNA and the current demand for high-volume testing, a less invasive, simpler to perform, and cheaper method to obtain DNA is desirable. We developed a method to obtain high-quality genomic DNA from buccal cells that has high acceptability and allows for a large number of PCR assays from a single sample. Sixty subjects vigorously swished 10 ml of undiluted commercial mouthwash in the mouth for 60 s and expelled the liquid into a collection container. DNA was isolated from the buccal cells with a rapid method using proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. Electrophoretic analysis of the extracted DNA showed detectable levels of high molecular weight genomic DNA in all samples. The DNA yields ranged from 0.2 to 134.0 microg, for an average of 49.7 microg. Using these samples, all 60 subjects were successfully genotyped by PCR-based assays for polymorphisms in the CYP1A1 (MspI and exon 7), CYP2E1 (RsaI), GSTM1, GSTT1, and NQO1 genes, confirming that the quality of DNA isolated from mouthwash samples was sufficient to reliably support PCR amplification. Storage of the (unprocessed) specimens at room temperature or at 37 degrees C for 1 week (temperature conditions that may be encountered when mailing samples) or at -20 degrees C for at least 6 months did not affect the DNA yield or ability to PCR amplify the samples. The results suggest that this mouthwash procedure may be suitable for large community-based studies of genetic susceptibility to disease in which samples can be collected by the participants themselves, mailed back to the study center, and stored for months prior to DNA analysis.
TL;DR: The finding of a close relationship among S, J, and Js genomes provides valuable markers for molecular cytogenetic analyses using S genome DNA probes to monitor the transfer of useful traits from Th.
Abstract: Genomic in situ hybridization (GISH) using genomic DNA probes from Thinopyrum elongatum (Host) D.R. Dewey (genome E, 2n = 14), Thinopyrum bessarabicum (Savul. & Rayss) A. Love (genome J, 2n = 14), and Pseudoroegneria strigosa (M. Bieb.) A. Love (genome S, 2n = 14), was used to examine the genomic constitution of Thinopyrum intermedium (Host) Barkworth & D.R. Dewey (2n = 6x = 42) and Thinopyrum ponticum (Podp.) Barkworth & D.R. Dewey (2n = 10x = 70). Evidence from GISH indicated that hexaploid Th, intermedium contained the J, Js, and S genomes, in which the J genome was related to the E genome of Th. elongatum and the J genome of Th. bessarabicum. The S genome was homologous to the S genome of Ps. strigosa, while the Js genome referred to modified J- or E-type chromosomes distinguished by the presence of S genome specific sequences close to the centromere. Decaploid Th. ponticum had only the two basic genomes J and Js. The Js genome present in Th. intermedium and Th. ponticum was homologous with E or J genomes, but was quite distinct at centromeric regions, which can strongly hybridize with the S genome DNA probe. Based on GISH results, the genomic formula of Th. intermedium was redesignated JJsS and that of Th. ponticum was redesignated JJJJsJs. The finding of a close relationship among S, J, and Js genomes provides valuable markers for molecular cytogenetic analyses using S genome DNA probes to monitor the transfer of useful traits from Th. intermedium and Th. ponticum to wheat.
TL;DR: The use of agarose blocks containing embedded DNA improves the PCR amplification from templates naturally contaminated with polysaccharides or humic acids, two powerful PCR inhibitors, overcoming this important problem of current amplification techniques.
Abstract: The use of agarose blocks containing embedded DNA improves the PCR amplification from templates naturally contaminated with polysaccharides or humic acids, two powerful PCR inhibitors. Presumably, the difference in size between the DNA macromolecules and these contaminants allows their effective removal from the agarose blocks by diffusion during the washing steps, whereas genomic DNA remains trapped within them. In addition, agarose-embedded DNA can be directly used for PCR since low melting point agarose does not interfere with the reaction. This simple and inexpensive method is also convenient for genomic DNAs extracted by other procedures, and it is potentially useful for samples containing other kinds of soluble inhibitors, overcoming this important problem of current amplification techniques.
TL;DR: This map provides a foundation for the study of the possible roles of ribosomal protein deficiencies in chromosomal and Mendelian disorders.
Abstract: We mapped 75 genes that collectively encode >90% of the proteins found in human ribosomes. Because localization of ribosomal protein genes (rp genes) is complicated by the existence of processed pseudogenes, multiple strategies were devised to identify PCR-detectable sequence-tagged sites (STSs) at introns. In some cases we exploited specific, pre-existing information about the intron/exon structure of a given human rp gene or its homolog in another vertebrate. When such information was unavailable, selection of PCR primer pairs was guided by general insights gleaned from analysis of all mammalian rp genes whose intron/exon structures have been published. For many genes, PCR amplification of introns was facilitated by use of YAC pool DNAs rather than total human genomic DNA as templates. We then assigned the rp gene STSs to individual human chromosomes by typing human‐rodent hybrid cell lines. The genes were placed more precisely on the physical map of the human genome by typing of radiation hybrids or screening YAC libraries. Fifty-one previously unmapped rp genes were localized, and 24 previously reported rp gene localizations were confirmed, refined, or corrected. Though functionally related and coordinately expressed, the 75 mapped genes are widely dispersed: Both sex chromosomes and at least 20 of the 22 autosomes carry one or more rp genes. Chromosome 19, known to have a high gene density, contains an unusually large number of rp genes (12). This map provides a foundation for the study of the possible roles of ribosomal protein deficiencies in chromosomal and Mendelian disorders. [The sequence data described in this paper have been submitted to GenBank. They are listed in Table 1.]
TL;DR: Using the recently introduced BigDye terminators, large-template DNA can be directly sequenced with custom primers on automated instruments without additional manipulations of template DNA, thereby bypassing tedious subcloning steps.
Abstract: In microbial genome or large-insert clone sequencing projects that use the predominant random subclone sequencing strategy, progress tends to decrease dramatically at late stages as one confronts gaps. At these points, DNA is under-represented or unstable in subclones (E.Y. Chen et al. 1996; Chissoe et al. 1997). Further sequencing with additional random subclones is then inefficient at best, and one must frequently employ alternative cloning systems or additional methods like long-range PCR to recover missing DNA (C.N. Chen et al. 1996). The variability of performance of these methods and the necessity for custom-tailored work tend to hamper the late stages of sequencing efforts. In contrast, if one can sequence directly from genomic DNA (or large-insert clones such as BACs or PACs) with walking primers, cumbersome work to fill gaps could be completed in a much shorter time.
As an example, in a recent project to sequence the 750-kb genome of Ureaplasma urealyticum (J. Glass, in prep.) assemblage of ∼13,000 sequence reads and combinatorial PCR reactions to join contigs left two gaps. No λ pUC, or M13 subclones were recovered that spanned the gaps, nor were PCR products derived with any of several sets of flanking primers. The difficulty of cloning these segments is probably attributable to repeated sequences in and near the two gaps, but the high sensitivity of the recently introduced BigDye terminator (Rosenblum et al. 1997) permitted direct sequencing of the gap regions on genomic U. urealyticum DNA templates. Using the conditions described in this report, two gaps of 259 and 121 bp were sequenced from both strands with walking primers to complete the project of 751,723 bp.
Direct sequencing was further tested for larger templates, and good results were reproducibly obtained with 1.2-Mb Mycoplasma fermentans, 2.3-Mb Streptococcus pneumoniae, and 4.6-Mb Escherichia coli genomic DNA (see example in Fig. Fig.1).1). In addition, several difficult gaps in sequencing projects with BAC clones, ranging in size from 140 to 250 kb, have also been filled in this manner. Essentially the method is applicable whenever 2–3 μg of high-quality large-template DNA is available.
Figure 1
Sequencing of E. coli K12 strain genomic DNA with BigDye terminators. Approximately 3 μg of E. coli DNA was sequenced with an apaG gene primer (5′-GTTCCCACACTCATTCATTA) using the conditions described in the text.
TL;DR: To facilitate analysis of the role of PSMA in carcinoma, the structural organization of the gene is determined and sequence differences between non-coding regions of the two genes suggests duplication and divergence occurred 22 million years ago.
TL;DR: A fosmid library with inserts containing approximately 40 kb of marine bacterial DNA (J. Stein, T. L. DeLong, J. Marsh, K. Y. Wu, H. Shizuya, and E. F.
Abstract: A fosmid library with inserts containing approximately 40 kb of marine bacterial DNA (J. L. Stein, T. L. Marsh, K. Y. Wu, H. Shizuya, and E. F. DeLong, J. Bacteriol. 178:591-599, 1996) yielded four clones with 16S rRNA genes from the order Planctomycetales. Three of the clones belong to the Pirellula group and one clone belongs to the Planctomyces group, based on phylogenetic and signature nucleotide analyses of full-length 16S rRNA genes. Sequence analysis of the ends of the genes revealed a consistent mismatch in a widely used bacterium-specific 16S rRNA PCR amplification priming site (27F), which has also been reported in some thermophiles and spirochetes.
TL;DR: A surprising network of metabolic functions is beginning to emerge in mature spermatozoa, which are normally repressed and are specifically activated upon exposure to appropriate stimuli.
Abstract: Summary Sperm cells from a variety of species share the spontaneous ability to take up foreign DNA. That feature has been exploited to generate genetically modified animals with variable efficiency in different species. An unexpectedly large set of factors appears to modulate the interaction of sperm cells with exogenous DNA. The binding is mediated by specific DNA-binding proteins and is antagonized by an inhibitory factor in the seminal fluid. A portion of sperm-bound DNA is internalized in nuclei, a process mediated by CD4 molecules. Sperm interaction with foreign DNA triggers endogenous nuclease(s) that cleaves both the exogenous and the genomic DNA, eventually leading to a cell death process which resembles apoptosis. Internalized foreign DNA sequences reach the nuclear matrix and undergo recombination with chromosomal DNA. From these studies, a surprising network of metabolic functions is beginning to emerge in mature spermatozoa, which are normally repressed and are specifically activated upon exposure to appropriate stimuli. BioEssays 20:955‐964, 1998. r 1998 John Wiley & Sons, Inc.