Inconsistencies between human genetic cytolocations and those derived using genomic sequence.

One result of the publishing of the human genome sequence is the ability to define objects through their position on the consensus sequence. While this has simplified the process of creating order maps for genes on a chromosome, it has created discrepancies between the published cytolocations of human genes, as presented through genetic references, and those locations derived computationally from the genomic sequence. For the 6,830 records with HUGO gene symbols shared between the online version of Mendelian Inheritance in Man and Ensembl, 18% of the records have a discrepancy of at least one cytogenetic band between the datasets. Discordance between data sets at this frequency would have a significant impact on the utility of datasets created by the amalgamation of numerous biological databases.

10th International Hugo mutation database initiative meeting, 19 April 2001, Edinburgh, Scotland.

The 10th International Mutation Database Initiative Meeting was held on April 19, 2001, in conjunction with the annual Human Genome Meeting in Edinburgh, Scotland. Key points of the meeting are described here. The BiSC WayStation was presented as an operational, viable beginning to the solution for the lack of centralized variation collection structures, with a number of possibilities, notably the BiSC Central Database and HGBASE, as candidates for storing the data. Exploration of new avenues of funding for this project, affiliation with Wiley-Liss, and the establishment of the Mutation Database Initiative (MDI) as a society were also discussed.

Influence of intercodon and base frequencies on codon usage in filarial parasites.

Base frequency, codon usage, and intercodon identity were analyzed in five filarial parasite species representing five Onchocercidae genera. Wucheria bancrofti, Brugia malayi, Onchocerca volvulus, Acanthocheilonema viteae, and Dirofilaria immitis gene sequences were downloaded from NCBI, and analysis was performed using locally designed computer programs and other freely available applications. A clear sequence bias was observed among the nematode species examined. At the nucleotide level, AT basepairs were present in gene sequences at higher frequencies than GC. In addition, codons ending in A or T were used proportionately more than those with G or C in the third-codon position. In addition, the amino acids used most often corresponded to codons ending in AT basepairs. Intercodon base proportion was biased in that A was found most often at N4, second only to T in certain specific cases. Since all of these sequence biases were observed in a relatively consistent fashion among all of the organisms studied, we conclude that sequence bias is a genetic characteristic, which is associated with multiple filarial genera.

TM Finder: a prediction program for transmembrane protein segments using a combination of hydrophobicity and nonpolar phase helicity scales.

Based on the principle of dual prediction by segment hydrophobicity and nonpolar phase helicity, in concert with imposed threshold values of these two parameters, we developed the automated prediction program TM Finder that can successfully locate most transmembrane (TM) segments in proteins. The program uses the results of experiments on a series of host-guest TM segment mimic peptides of prototypic sequence KK AAAXAAAAAXAAWAAXAAAKKKK-amide (where X = each of the 20 commonly occurring amino acids) through which an HPLC-derived hydropathy scale, a hydrophobicity threshold for spontaneous membrane insertion, and a nonpolar phase helical propensity scale were determined. Using these scales, the optimized prediction algorithm of TM Finder defines TM segments by first searching for competent core segments using the combination of hydrophobicity and helicity scales, and then performs a gap-joining operation, which minimizes prediction bias caused by local hydrophilic residues and/or the choice of window size. In addition, the hydrophobicity threshold requirement enables TM Finder to distinguish reliably between membrane proteins and globular proteins, thereby adding an important dimension to the program. A full web version of the TM Finder program can be accessed at http://www.bioinformatics-canada.org/TM/.

Human mapping databases.

This unit concentrates on the data contained within two human genome databasesGDB (Genome Database) and OMIM (Online Mendelian Inheritance in Man)and includes discussion of different methods for submitting and accessing data. An understanding of electronic mail, FTP, and the use of a World Wide Web (WWW) navigational tool such as Netscape or Internet Explorer is a prerequisite for utilizing the information in this unit.

Recent advances in bioinformatics in the medical research environment and applications to the study of skin diseases.

BACKGROUND: The computer has become increasingly intertwined in society for the past 30 years. Within the academic health science centre, there is an increasing need for researchers to become skilled at using the Internet as a mechanism for the retrieval of scientific results and the underlying data. The discipline of bioinformatics, which uses computer technology to provide answers to biological questions, has been expanding in scope and utility for the past decade. Increasing numbers of research groups have been investing in bioinformatics infrastructure to aid in the research process. These continuing investments have led to the establishment for the first time of a supercomputing facility within a hospital. Such computational power is being used for the mapping of genes and the study of human disease. OBJECTIVE: A discussion of the increasing role of computational biology in the research environment of the clinician scientist is presented here. CONCLUSIONS: Though the investment in a supercomputer may not be possible in most research settings, several less expensive alternatives relying on existing desktop computers can provide supercomputer-like performance within nearly any environment.

Central mutation databases--a review.

The Internet has been a key component in the coordination of the diverse group of scientists involved in the Human Genome Project. Nowhere has this contribution been more critical than in the maintenance and exchange of information about genetic variation and mutation. Whereas the majority of DNA sequence is generated and stored by a relatively few sites, a far greater number of researchers investigate the variations in that sequence from sites scattered worldwide. It falls to central databases to utilize the Internet to assemble data from these sites and make them available to the greater human genomic community.

Future vision of the GDB human genome database.

In 1973, scientists assembled at the first Human Gene Mapping Workshop to discuss the 64 human genes mapped at that time. In 1989, the GDB Human Genome Database was created to store information on 1, 700 mapped human genes. Ten years later, as the human genome project closes in on the release of the complete DNA sequence holding as many as 100,000 human genes, GDB is evolving to continue to meet the needs of the scientific community. Well known as a resource for data which has been stringently reviewed as part of the curation process, GDB prepares to continue to provide a compilation of the human genome including maps, map objects, polymorphisms, and mutations. As more sites across the Internet are established to share biological information, it becomes increasingly burdensome for the scientist to collect data from all sources of a particular domain. In an attempt to reduce this burden, GDB continues to load data from large genome centres and accept submissions from researchers around the world. Moreover, GDB looks to provide a mechanism to link gene-related information to the human reference sequence. In doing this, GDB plans to establish federated linkages with "boutique" databases around the world that could contain enormous amounts of valuable information about specific genes or chromosomes.

Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection.

Recent evidence suggests that infertile males donating semen for intracytoplasmic sperm injection (ICSI) may be at an increased risk of transmitting numerical (predominantly sex chromosome) abnormalities to their offspring. The present study was designed to determine aneuploidy in spermatozoa from oligoasthenoteratozoospermic (OAT) patients undergoing ICSI. Aneuploidy frequencies of 12 autosomes and the sex chromosomes were determined by fluorescence in-situ hybridization (FISH) on spermatozoa from fresh ejaculate of nine severe OAT patients and four proven fertile donors. FISH, using directly labelled (fluorochrome-dUTP) satellite or contig DNA probes specific for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X, and Y, was performed on decondensed spermatozoa. Per chromosome disomy frequencies for autosomes and sex chomosomes in OAT males were 0-5. 4%. In contrast, the disomy frequencies in controls were 0.05-0.2%. The frequency of diploid spermatozoa in OAT patients was 0.4-9.6%; controls showed a mean of 0.04%. Using recently developed formulae, the total aneuploidy in our OAT patient population was estimated to be 33-74%. In contrast, estimates of mean total aneuploidy in the spermatozoa of controls ranged from 4.1 to 7.7%, depending upon method of calculation. Six series of ICSI were performed on five of the OAT patients. Four resulted in no establishment of pregnancy; the others failed to establish ongoing pregnancies. Our cytogenetic data show significantly elevated frequencies of diploidy, autosomal disomy and nullisomy, sex chromosome aneuploidy, and total aneuploidy in OAT patients, which may contribute to the patients' infertility.

The GDB Human Genome Data Base anno 1994.

In 1991 the Genome Data Base at Johns Hopkins University School of Medicine was selected as the central repository for mapping data from the Human Genome Project, and was funded by NIH and DOE under a three year award. GDB has now finished 28 months of Federally funded operation. During this period a great deal of progress and many internal changes have taken place. In addition, many changes have also occurred in the external environment, and GDB has adapted its strategies to play an appropriate role in those changes as well. Recognizing the central role of mapping information in the genome project, it is important that GDB respond aggressively to the increasing demands of genomic researchers, as well as formulate a program of response to a number of long standing, but still unmet, needs of that community. It is even more important that GDB provide leadership in the genome informatics enterprise. Three themes described here are dominant in our future plans and represent the essence of the major changes made in the past year. They include: enhanced data acquisition, better map representation, and full integration into the collection of genomic databases.

The GDB human genome data base anno 1993.

Version 5.0 of the Genome Data Base (GDB) was released in March 1993. This document describes some of the significant changes to the types of data which are stored within the GDB. In addition to handling a wider scope of data, the GDB 5.0 application software now supports the X-Windows protocol. Although the GDB software still remains the most widely utilized method for accessing the data, alternate methods of access are now available, including direct SQL (Structured Query Language) queries, FTP (Internet File Transfer Protocol), WAIS (Wide Area Information Server), and other tools produced by third-party developers.

PCAP: probe choice and analysis package--a set of programs to aid in choosing synthetic oligomers for contig mapping.

In the program, PCAP, we provide a methodology for choosing synthetic oligonucleotide probes to be used in contig mapping experiments. The package serves the purpose of presenting a series of short oligonucleotides (8-12mers) that are chosen based on constraints with respect to frequency of occurrence within a particular genome and the G+C content of the oligonucleotides. The four programs contained within the package: (i) convert GenBank files to a format useable by the package; (ii) calculate trinucleotide and tetranucleotide frequencies in available sequence data on a particular species; (iii) present the user with upper and lower bounds on the frequencies of hybridization sites for oligonucleotide probes of length 8-12, (iv) allow the user to place constraints on site frequency and G+C content and provides a list of short probe sequences that fit these criteria. These sequences can then be synthetically produced and used in hybridization experiments to carry out contig mapping.

ODS: ordering DNA sequences--a physical mapping algorithm based on simulated annealing.

In the program ODS we provide a methodology for quickly ordering random clones into a physical map. The process of ordering individual clones with respect to their position along a chromosome is based on the similarity of binary signatures assigned to each clone. This binary signature is obtained by hybridizing each clone to a panel of oligonucleotide probes. By using the fact that the amount of overlap between any two clones is reflected in the similarity of their binary signatures, it is possible to reconstruct a chromosome by minimizing the sum of linking distances between an ordered sequence of clones. Unlike other programs for physical mapping, ODS is very general in the types of data that can be utilized for chromosome reconstruction. Any trait that can be scored in a presence--absence manner, such as hybridized synthetic oligonucleotides, restriction endonuclease recognition sites or single copy landmarks, can be used for analysis. Furthermore, the computational requirements for the construction of large physical maps can be measured in a matter of hours on work-stations such as the VAX2000.

CMAP: contig mapping and analysis package, a relational database for chromosome reconstruction.

In the contig mapping and analysis package, CMAP, we provide a foundation for reverse genetics by organizing information about DNA fragments obtained from an organism's genome into a physical map. The user can store information about a particular segment of DNA. This information can be both descriptive, such as any genes contained in a particular DNA fragment, or experimental, such as hybridization profiles or restriction digest patterns for comparison with other fragments. The package can then be instructed to update the physical map or provide information on a DNA fragment within the map, such as its location. The user interface is designed to minimize the learning curve associated with database usage, while eliminating the possibility of entering data outside the ranges of fields through error-checking protocols. Queries are currently accomplished by the use of dynamic SQL (structured query language), which gives the user the ability to build queries based on any combination of the attributes contained within the database without requiring that all possible queries be permanently programmed within the query software. In order to eliminate the need for knowledge of SQL, an interface was designed to allow users to build queries by menu choices. Thus, CMAP is a software package supporting a database for both the production and storage of a physical map as well as being the first step toward the production of a physical mapping workstation.

The use of simulated annealing in chromosome reconstruction experiments based on binary scoring.

We present a method of combinatorial optimization, simulated annealing, to order clones in a library with respect to their position along a chromosome. This ordering method relies on scoring each clone for the presence or absence of specific target sequences, thereby assigning a digital signature to each clone. Specifically, we consider the hybridization of oligonucleotide probes to a clone to constitute the signature. In that the degree of clonal overlap is reflected in the similarity of their signatures, it is possible to construct maps based on the minimization of the differences in signatures across a reconstructed chromosome. Our simulations show that with as few as 30 probes and a clonal density of 4.5 genome equivalents, it is possible to assemble a small eukaryotic chromosome into 33 contiguous blocks of clones (contigs). With higher clonal densities and more probes, this number can be reduced to less than 5 contigs per chromosome.

The application of Markov chain analysis to oligonucleotide frequency prediction and physical mapping of Drosophila melanogaster.

Here we compare several methods for predicting oligonucleotide frequencies in 691 kb of Drosophila melanogaster DNA. As in previous work on Escherichia coli and Saccharomyces cerevisiae, a relatively simple equation based on tetranucleotide frequencies can be used in predicting frequencies of higher order oligonucleotides. For example, the mean of observed/expected abundances of 4,096 hexamers was 1.07 with a sample standard deviation of .55. This simple predictor arises by considering each base on the sense strand of D. melanogaster to depend only on the three bases 5' to it (a 3rd order Markov chain) and is more accurate than the random predictor. This equation is useful in predicting restriction enzyme fragment sizes, selecting restriction enzymes that cut preferentially in coding vs noncoding regions, and in selecting probes to fingerprint clones in contig mapping. Once again, this equation well predicts the occurrence of higher order oligonucleotides, supporting our hypothesis that this predictor holds in evolutionarily diverse organisms. When ranked from highest to lowest abundance, the observed frequencies of oligomers of a given length are closely tracked by the predicted abundances of a 3rd order Markov chain. Through use of the dependence of oligomer frequencies on base composition, we report a list of oligomers that will be useful for the completion of a cosmid physical map of D. melanogaster. Presently, the library is such that it will be possible to construct large contigs using only 30 oligonucleotide probes to fingerprint cosmids.

Chromosome-specific recombinant DNA libraries from the fungus Aspergillus nidulans.

Development of physical genomic maps is facilitated by identification of overlapping recombinant DNA clones containing long chromosomal DNA inserts. To simplify the analysis required to determine which clones in a genomic library overlap one another, we partitioned Aspergillus nidulans cosmid libraries into chromosome-specific subcollections. The eight A. nidulans chromosomes were resolved by pulsed field gel electrophoresis and hybridized to filter replicas of cosmid libraries. The subcollections obtained appeared to be representative of the chromosomes based on the correspondence between subcollection size and chromosome length. A sufficient number of clones was obtained in each chromosome-specific subcollection to predict the overlap and assembly of individual clones into a limited number of contiguous regions. This approach should be applicable to many organisms whose genomes can be resolved by pulsed field gel electrophoresis.