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Biomedical subjects

R Drmanac

Publications and source records attributed to R Drmanac.

At least 19 recordsLinked to original sources
PubMed1999

Cloning of a novel epidermal growth factor repeat containing gene EGFL6: expressed in tumor and fetal tissues.

The epidermal growth factor (EGF) repeat superfamily of genes often encodes proteins that govern cellular proliferative responses. Using a high-throughput screening by hybridization approach, a novel human EGF repeat superfamily member that maps to human chromosome X was identified. Termed EGFL6, the gene encodes a predicted signal peptide, suggesting that it is secreted. Other predicted features include four and one-half EGF-like repeat domains, two N-linked glycosylation sites, an integrin association motif (RGD), and a tyrosine phosphorylation site. Importantly, its transcripts are expressed in brain and lung tumor and fetal tissues, but are generally absent from normal adult tissues. Implications with respect to cell cycle regulation and oncogenesis are discussed.

Adult↗
PubMed1999

IL1HY1: A novel interleukin-1 receptor antagonist gene.

Interleukin-1 is a potent mediator of inflammation, involved in regulating a wide variety of physiological and cellular events. We have identified and characterized a novel member of the human interleukin-1 gene family (IL1HY1). The encoded protein demonstrates significant amino acid homology to the receptor antagonist (IL-1ra) at 52%. The gene was mapped to the long arm of chromosome 2, in close proximity to the IL-1 locus. IL1HY1 message is tightly regulated being most predominantly expressed in the skin, but also detected in the spleen, brain leukocyte, and macrophage cell types. Furthermore, the message can be induced in THP-1 cells by phorbol ester (PMA) and lipopolysaccharide (LPS) treatment.

Amino Acid Sequence↗
PubMed1998

Accurate sequencing by hybridization for DNA diagnostics and individual genomics.

Medical DNA diagnostics will increasingly rely on an accurate and inexpensive identification of mutations that affect the function of a gene. To validate diagnostic sequencing by hybridization (SBH), a number of p53 samples were analyzed with the complete set of 8192 noncomplementary 7-mer oligonucleotides. In four repeated, blind experiments we accurately sequenced 1.1 kb per each of 12 homozygote and heterozygote samples possessing base substitutions, insertions, and deletions. This SBH variant offers a high throughput platform to inexpensively sequence individual gene or pathogen genome samples within the clinical laboratory setting.

DNA↗
PubMed1996

Gene-representing cDNA clusters defined by hybridization of 57,419 clones from infant brain libraries with short oligonucleotide probes.

Diverse biochemical and computational procedures and facilities have been developed to hybridize thousands of DNA clones with short oligonucleotide probes and subsequently to extract valuable genetic information. This technology has been applied to 73,536 cDNA clones from infant brain libraries. By a mutual comparison of 57,419 samples that were successfully scored by 200-320 probes, 19,726 genes have been identified and sorted by their expression levels. The data indicate that an additional 20,000 or more genes may be expressed in the infant brain. Representative clones of the found genes create a valuable resource for complete sequencing and functional studies of many novel genes. These results demonstrate the unique capacity of hybridization technology to identify weakly transcribed genes and to study gene networks involved in organismal development, aging, or tumorigenesis by monitoring the expression of every gene in related tissues, whether known or still undiscovered.

Base Sequence↗
PubMed1994

Processing of cDNA and genomic kilobase-size clones for massive screening, mapping and sequencing by hybridization.

Efficient procedures for managing a large number of M13 or plasmid clones have been developed. In addition to picking, clones are directly arrayed in multiwell plates by dispensing diluted transformation mixtures. Metal pin arrays are used for fast inoculations of preparative plates filled by medium or by PCR mixture. Growth of M13 clones in multiwell plates is optimized to obtain a consistently high yield, and a PCR protocol is defined for reliable amplification of several thousand M13 or plasmid inserts per day in BioOvens. Over 80,000 cDNA inserts have been amplified. The phages or amplified inserts are spotted on nylon filters using an array of pins having a flat bottom, 0.3 mm in diameter. The procedures are suitable for an automated processing of hundreds of thousands of short clones from representative cDNA and genomic libraries. Hybridization of arrayed clones with oligonucleotide and complex probes can simplify the search for new genes and accelerate large-scale sequencing.

Bacteriophage M13↗
PubMed1993

DNA sequence determination by hybridization: a strategy for efficient large-scale sequencing.

The concept of sequencing by hybridization (SBH) makes use of an array of all possible n-nucleotide oligomers (n-mers) to identify n-mers present in an unknown DNA sequence. Computational approaches can then be used to assemble the complete sequence. As a validation of this concept, the sequences of three DNA fragments, 343 base pairs in length, were determined with octamer oligonucleotides. Possible applications of SBH include physical mapping (ordering) of overlapping DNA clones, sequence checking, DNA fingerprinting comparisons of normal and disease-causing genes, and the identification of DNA fragments with particular sequence motifs in complementary DNA and genomic libraries. The SBH techniques may accelerate the mapping and sequencing phases of the human genome project.

Animals↗
PubMed1992

Sequencing by hybridization: towards an automated sequencing of one million M13 clones arrayed on membranes.

An immediately applicable variant of the sequencing by hybridization (SBH) method is under development with the capacity to determine up to 100 million base pairs per year. The proposed method comprises six steps: (i) arraying genomic or cDNA M13 clones in 864-well plates (wells of 2 mm); (ii) preparation of DNA samples for spotting by growth of the M13 clones or by polymerase chain reaction (PCR) of the inserts using standard 96-well plates, or plates having as many as 864 correspondingly smaller wells; (iii) robotic spotting of 13,824 samples on an 8 x 12 cm nylon membrane, or correspondingly more, on up to 6 times larger filters, by offset printing with a 96 or 864 0.4 mm pin device; (iv) hybridization of dotted samples with 200-2000 32P-labeled probes comprising 16-256 10-mers having a common 8-mer, 7-mer, or 6-mer in the middle (20 probes per day, each hybridized with 250,000 dots); (v) scoring hybridization signals of 5 million sample-probe pairs per day using storage phosphor plates; and (vi) computing clone order and partial-to-complete DNA sequences using various heuristic algorithms. Genome sequencing based on a combination of this method and gel sequencing techniques may be significantly more economical than gel methods alone.

Bacteriophage M13↗
PubMed1991

DNA sequencing by hybridization: 100 bases read by a non-gel-based method.

Determination of the sequences of human and other complex genomes requires much faster and less expensive sequencing processes than the methods in use today. Sequencing by hybridization is potentially such a process. In this paper we present hybridization data sufficient to accurately read a known sequence of 100 base pairs. In independent reactions, octamer and nonamer oligonucleotides derived from the sequence hybridized more strongly to this DNA than to controls. The 93 consecutive overlapping probes were derived from a 100-base-pair segment of test DNA and additional probes were generated by incorporation of a noncomplementary base at one of the ends of 12 of the basic probes. These 12 additional probes also had a full-match target in one of the control DNAs. The test and one of five control DNAs spotted on nylon filters were hybridized with 83 octamers and 22 nonamers under low-temperature conditions. A stronger signal in DNA containing a full-match target compared to DNA with only mismatched targets was obtained with all 105 probes. In 3 cases (2.9%), the difference of signals was not significant (less than 2-fold) due to inefficient hybridization and the consequently higher influence of background. The hybridization pattern obtained enabled us to resequence the 100 base pairs by applying an algorithm that tolerates an error rate much higher than was observed in the experiment. With this result, the technological components of large-scale DNA sequencing using the sequencing by hybridization method are in place.

Base Sequence↗
PubMed1991

W (A or T) sequences as probes and primers suitable for genomic mapping and fingerprinting.

A limitation to the use of oligonucleotide probes as tools for genetic and physical mapping has been the low hybridization positive frequency obtained by oligonucleotides of sufficient length to hybridize preferentially to cloned insert DNA (and not host E. coli genomic DNA). Both computer and experimental results now indicate that oligonucleotide probes composed of W (A or T) sequence are preferentially found in eukaryotic DNA, and can be used to provide high frequency, discriminative hybridization. Such W sequences may be useful as either probes or PCR primers in molecular diagnostic applications as well as in genetic and physical mapping.

Adenine↗
PubMed1991

An algorithm for the DNA sequence generation from k-tuple word contents of the minimal number of random fragments.

An algorithm is described for generation of the long sequence written in a four letter alphabet from the constituent k-tuple words in the minimal number of separate, randomly defined fragments of the starting sequence. It is primarily intended for use in sequencing by hybridization (SBH) process- a potential method for sequencing human genome DNA (Drmanac et al., Genomics 4, pp. 114-128, 1989). The algorithm is based on the formerly defined rules and informative entities of the linear sequence. The algorithm requires neither knowledge on the number of appearances of a given k-tuple in sequence fragments, nor the information on which k-tuple words are on the ends of a fragment. It operates with the mixed content of k-tuples of the various lengths. The concept of the algorithm enables operations with the k-tuple sets containing false positive and false negative k-tuples. The content of the false k-tuples primarily affects the completeness of the generated sequence, and its correctness in the specific cases only. The algorithm can be used for the optimization of SBH parameters in the simulation experiments, as well as for the sequence generation in the real SBH experiments on the genomic DNA.

Algorithms↗
PubMed1990

Reliable hybridization of oligonucleotides as short as six nucleotides.

Although there are many new applications for hybridizing short, synthetic oligonucleotide probes to DNA, such applications have not included determining unknown sequences of DNA. The lack of clear discrimination in hybridization of oligo probes shorter than 11 nucleotides and the lack of a theoretical understanding of factors influencing hybridization of short oligos have hampered the development of their use. We have found conditions for reliable hybridization of oligonucleotides as short as seven nucleotides to cloned DNA or to oligonucleotides attached to filters. Low-temperature hybridization and washing conditions, in contrast to the high stringency conditions currently used in hybridization experiments, have the potential for allowing the simple use of all oligos of six nucleotides or longer in meaningful hybridizations. We also present the hybridization discrimination theory that provides the conceptual framework for understanding these results.

DNA Fingerprinting↗
PubMed1990

Origin of rat beta-globin haplotypes containing three and five genes.

We have reported in rat three adult beta-gene haplotypes containing either five or three genes. Detailed sequence analysis reveals that the leftmost gene is the major gene and that at the opposite end downstream lies the minor gene. All of the genes lying between them are minor-major hybrids indicating their origin by unequal crossing-over. In two haplotypes beta-globin genes were found with an L1(1) element inserted directly into IVS2. The described results allow the formulation of a pathway of mutational events leading from the ancient two-beta-gene rodent ancestor through a three-gene haplotype to five-gene haplotypes, one of which is postulated to have arisen in common laboratory strains since their capture in the wild.

Animals↗
PubMed1989

Variant chromosomal arrangement of adult beta-globin genes in rat.

The genomic organization of three haplotypes of beta-globin genes was determined to resolve the question of the number of those genes in rat. Haplotype a, found in inbred strain DA, has three genes or pseudogenes, while haplotypes b, found in AO, Y5 and Wistar strains, and c, found in Wistar strain, have five genes or pseudogenes each. In haplotypes b and c, the first gene is of beta major type and the remaining four are of beta minor type. Partial sequencing of six out of 13 genes shows that duplications of beta minor genes are causing polymorphism in a number of genes. Also, in haplotype b two beta minor genes have a 6.5-kb intron 2, while in haplotype c only one beta minor gene contains such a large intron 2. The three structurally different haplotypes described are not interconvertible by single recombination events. The results indicate that the rat has the highest number of adult beta-globin genes found in mammals so far.

Amino Acid Sequence↗