Integration of cytogenetic landmarks into the draft sequence of the human genome.

We have placed 7,600 cytogenetically defined landmarks on the draft sequence of the human genome to help with the characterization of genes altered by gross chromosomal aberrations that cause human disease. The landmarks are large-insert clones mapped to chromosome bands by fluorescence in situ hybridization. Each clone contains a sequence tag that is positioned on the genomic sequence. This genome-wide set of sequence-anchored clones allows structural and functional analyses of the genome. This resource represents the first comprehensive integration of cytogenetic, radiation hybrid, linkage and sequence maps of the human genome; provides an independent validation of the sequence map and framework for contig order and orientation; surveys the genome for large-scale duplications, which are likely to require special attention during sequence assembly; and allows a stringent assessment of sequence differences between the dark and light bands of chromosomes. It also provides insight into large-scale chromatin structure and the evolution of chromosomes and gene families and will accelerate our understanding of the molecular bases of human disease and cancer.

dbSNP: the NCBI database of genetic variation.

In response to a need for a general catalog of genome variation to address the large-scale sampling designs required by association studies, gene mapping and evolutionary biology, the National Center for Biotechnology Information (NCBI) has established the dbSNP database [S.T.Sherry, M.Ward and K. Sirotkin (1999) Genome Res., 9, 677-679]. Submissions to dbSNP will be integrated with other sources of information at NCBI such as GenBank, PubMed, LocusLink and the Human Genome Project data. The complete contents of dbSNP are available to the public at website: http://www.ncbi.nlm.nih.gov/SNP. The complete contents of dbSNP can also be downloaded in multiple formats via anonymous FTP at ftp://ncbi.nlm.nih.gov/snp/.

dbSNP: a database of single nucleotide polymorphisms.

In response to a need for a general catalog of genome variation to address the large-scale sampling designs required by association studies, gene mapping and evolutionary biology, the National Cancer for Biotechnology Information (NCBI) has established the dbSNP database. Submissions to dbSNP will be integrated with other sources of information at NCBI such as GenBank, PubMed, LocusLink and the Human Genome Project data. The complete contents of dbSNP are available to the public at website: http://www.ncbi.nlm.nih.gov/SNP. Submitted SNPs can also be downloaded via anonymous FTP at ftp://ncbi.nlm.nih.gov/snp/

Use of molecular variation in the NCBI dbSNP database.

While high quality information regarding variation in genes is currently available in locus-specific or specialized mutation databases, the need remains for a general catalog of genome variation to address the large-scale sampling designs required by association studies, gene mapping, and evolutionary biology. In response to this need, the National Center for Biotechnology Information (NCBI) has established the dbSNP database http://ncbi. nlm.nih.gov/SNP/ to serve as a generalized, central variation database. Submissions to dbSNP will be integrated with other sources of information at NCBI such as GenBank, PubMed, LocusLink, and the Human Genome Project data, and the complete contents of dbSNP are available to the public via anonymous FTP. Hum Mutat 15:68-75, 2000. Published 2000 Wiley-Liss, Inc.

Determination of eukaryotic protein coding regions using neural networks and information theory.

Our previous work applied neural network techniques to the problem of discriminating open reading frame (ORF) sequences taken from introns versus exons. The method counted the codon frequencies in an ORF of a specified length, and then used this codon frequency representation of DNA fragments to train a neural net (essentially a Perceptron with a sigmoidal, or "soft step function", output) to perform this discrimination. After training, the network was then applied to a disjoint "predict" set of data to assess accuracy. The resulting accuracy in our previous work was 98.4%, exceeding accuracies reported in the literature at that time for other algorithms. Here, we report even higher accuracies stemming from calculations of mutual information (a correlation measure) of spatially separated codons in exons, and in introns. Significant mutual information exists in exons, but not in introns, between adjacent codons. This suggests that dicodon frequencies of adjacent codons are important for intron/exon discrimination. We report that accuracies obtained using a neural net trained on the frequency of dicodons is significantly higher at smaller fragment lengths than even our original results using codon frequencies, which were already higher than simple statistical methods that also used codon frequencies. We also report accuracies obtained from including codon and dicodon statistics in all six reading frames, i.e. the three frames on the original and complement strand. Inclusion of six-frame statistics increases the accuracy still further. We also compare these neural net results to a Bayesian statistical prediction method that assumes independent codon frequencies in each position. The performance of the Bayesian scheme is poorer than any of the neural based schemes, however many methods reported in the literature either explicitly, or implicitly, use this method. Specifically, Bayesian prediction schemes based on codon frequencies achieve 90.9% accuracy on 90 codon ORFs, while our best neural net scheme reaches 99.4% accuracy on 60 codon ORFs. "Accuracy" is defined as the average of the exon and intron sensitivities. Achievement of sufficiently high accuracies on short fragment lengths can be useful in providing a computational means of finding coding regions in unannotated DNA sequences such as those arising from the mega-base sequencing efforts of the Human Genome Project. We caution that the high accuracies reported here do not represent a complete solution to the problem of identifying exons in "raw" base sequences. The accuracies are considerably lower from exons of small length, although still higher than accuracies reported in the literature for other methods. Short exon lengths are not uncommon.(ABSTRACT TRUNCATED AT 400 WORDS)

The distribution of interspersed repetitive DNA sequences in the human genome.

The distribution of interspersed repetitive DNA sequences in the human genome has been investigated, using a combination of biochemical, cytological, computational, and recombinant DNA approaches. "Low-resolution" biochemical experiments indicate that the general distribution of repetitive sequences in human DNA can be adequately described by models that assume a random spacing, with an average distance of 3 kb. A detailed "high-resolution" map of the repetitive sequence organization along 400 kb of cloned human DNA, including 150 kb of DNA fragments isolated for this study, is consistent with this general distribution pattern. However, a higher frequency of spacing distances greater than 9.5 kb was observed in this genomic DNA sample. While the overall repetitive sequence distribution is best described by models that assume a random distribution, an analysis of the distribution of Alu repetitive sequences appearing in the GenBank sequence database indicates that there are local domains with varying Alu placement densities. In situ hybridization to human metaphase chromosomes indicates that local density domains for Alu placement can be observed cytologically. Centric heterochromatin regions, in particular, are at least 50-fold underrepresented in Alu sequences. The observed distribution for repetitive sequences in human DNA is the expected result for sequences that transpose throughout the genome, with local regions of "preference" or "exclusion" for integration.

A computer program to display codon changes caused by mutagenesis.

A FORTRAN program for displaying the correspondence between codon changes and different possible base changes is presented. Changes of both single bases and dimers are considered. The user can specify the mutagenesis spectrum. Additionally, the user can choose whether or not to consider single or double events in a codon and whether or not to consider the possibility that the change of two bases (a dimer) can overlap a codon boundary. Furthermore, a variety of ways may be chosen to display and summarize the codon changes that can result from the specified mutagenesis. A user-supplied sequence or the genetic code table can be analyzed.

Bacteriophage T2 and T4, dam+ and damh and Eco dam+ methylation: preference at different sites.

We present a method for determining preference for methylation at minor methylation sites. The target DNA sequence is first subjected to computer-assisted analysis to predict which restriction endonuclease(s) will generate fragments that will contain only one or two likely minor methylation site(s). The target DNA is then methylated in vitro with a radioactive methyl-group donor and subjected to digestion by the chosen restriction enzyme(s). The amount of radioactivity in the various fragments is determined, after separating them using polyacrylamide gel electrophoresis. We documented the effect of nearby bases on the methylation preference and the relative preference for methylation at some specific minor methylation sites.

The autodegradation of deoxyribonucleic acid (DNA) in human rib bone and its relationship to the time interval since death.

This research explored the feasibility of using the degradation rate of deoxyribonucleic acid (DNA) in human rib bone to determine the time interval since death. Postmortem human rib samples were surface sterilized and incubated under sterile conditions in either high or low humidity conditions at room temperature for a period of weeks. At selected times, portions of the bone were cut away, and the DNA from these samples was extracted and subjected to strand separating gel electrophoresis. The DNAs in the gels were transferred to a nylon membrane, preserving their relative positions as in the gel, and probed with radioactive total genomic human DNA. Autoradiograms produced were scanned and digitized. When the samples were incubated under identical conditions, the degradation rate of DNA in samples from different individuals appeared very similar. The DNA degradation rate may vary with temperature and humidity more than it varies between individuals.

Quantitating small volumes of dilute DNA samples containing sodium dodecyl sulfate.

A technique to quantitate small volumes of dilute solutions of different-sized DNA fragments has been developed. The detection limit was 0.7 micrograms/ml and the technique could be used even in the presence of diffusable substances, including those such as sodium dodecyl sulfate which affect surface tension and also exhibit fluorescence when stained with ethidium bromide and excited by ultraviolet light. The DNA was mixed with low-melting-point agarose and pipetted into preformed wells in an agarose plate, where it solidified. After diffusion of small molecules, the amount of DNA was estimated by comparing ethidium bromide-mediated fluorescence of samples with that of standards.

A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum.

By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).

Advantages to mutagenesis techniques generating populations containing the complete spectrum of single codon changes.

The limitations of current mutagenesis techniques are analyzed in terms of the number and kinds of codon changes they make and in terms of the population size needed to produce all single or multiple amino acid variants. It is shown how a technique that can alter a single codon of a gene, producing all possible variant codons without affecting the rest of the gene, has certain advantages, if it can be used at each place in the gene in one experiment. Such a technique has advantages when the goals are to understand: (1) how specific structural alterations in a mutant protein cause it to function in a different but specific way, (2) how to predict which amino acids in a protein contact or interact with each other, and (3) why a protein is more or less sensitive to mutational disruption, depending upon the specific mutation. This is because it would generate the maximum number of (1) mutant proteins with different functions, (2) intracistronic suppressor for any starting mutation, and (3) random amino acid substitutions at random places. Furthermore, such a technique could produce useful variants more quickly and on a smaller scale than either evolution or current methods.

Deletion polymorphism in a Drosophila melanogaster heat shock gene.

We have continued the transcriptional analysis of the region of cytological locus 67B that contains the four small heat shock genes and other genes. Transcription from one of the heat shock genes in the region, hsp 26, takes place during high temperature treatment and at certain developmental stages, without heat shock, in several tissues, such as imaginal discs and adult ovaries. Observations of unexpected products after nuclease protection experiments provided the first indication of what genomic blot experiments showed to be small deletions. The alleles containing the deletion are expressed at the same level as the wild type allele. The deletion shortens the protein product, implying that it is in the coding region. Furthermore, flies homozygous for one of the deletion alleles are viable.

Kinetic control of the length of very short homopolymeric additions by terminal deoxynucleotidyltransferase.

This work is an investigation of the practicality of kinetic control of the length of very short deoxynucleotide homopolymeric additions by terminal deoxynucleotidyltransferase. For such very short additions, the possibility that terminal deoxynucleotidyltransferase acts differently with each deoxytriphosphate, or shows interaction effects when presented with multiple deoxytriphosphates, was investigated. Different relative rates of priming and different relative rates of subsequent additions were found for each deoxytriphosphate. Each triphosphate reacted uniquely, and one case of interaction was found, with adenosine interfering with cytidine addition.

Isolation and characterization of the DNA region encoding nodulation functions in Bradyrhizobium japonicum.

The DNA region encoding early nodulation functions of Bradyrhizobium japonicum 3I1b110 (I110) was isolated by its homology to the functionally similar region from Rhizobium meliloti. Isolation of a number of overlapping recombinant clones from this region allowed the construction of a restriction map of the region. The identified nodulation region of B. japonicum shows homology exclusively to those regions of R. meliloti and Rhizobium leguminosarum DNA known to encode early nodulation functions. The region of homology with these two fast-growing Rhizobium species was narrowed to an 11.7-kilobase segment. A nodulation-defective mutant of Rhizobium fredii USDA 201, strain A05B-2, was isolated and found to be defective in the ability to curl soybean root hairs. Some of the isolated recombinant DNA clones of B. japonicum were found to restore wild-type nodulation function to this mutant. Analysis of the complementation results allows the identification of a 1.8-kilobase region as essential for restoration of Hac function.

Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples.

The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations.

Repetitive DNA of Candida albicans: nuclear and mitochondrial components.

We report the isolation and analysis of the rapidly reassociating DNA of the pathogenic, dimorphic fungus Candida albicans. Minicot analysis of whole-cell repetitive DNA suggested that a significant portion of this component was mitochondrial DNA. Genomic blot hybridizations in which radioactive whole-cell repetitive DNA was used as a probe revealed eight major EcoRI bands in the molecular weight range resolved by the gel system used. Isolation and analysis of high-purity mitochondrial DNA have shown that five of these bands are of mitochondrial origin. The remaining three bands are of nuclear origin and represent repetitive sequences that are found in the nuclear genome. Attempts to isolate nuclear DNA that was completely free of mitochondrial DNA contamination were unsuccessful.