Complementary DNA sequencing: expressed sequence tags and human genome project.

Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.

Evidence for lipopolysaccharide binding in human granulosa-luteal cells.

We investigated whether human granulosa-luteal (GL) cells exhibited lipopolysaccharide (LPS)-binding protein, and the response of follicular aspirate cells to LPS in vitro. Follicular aspirates taken from a human in-vitro fertilization and gamete intra-fallopian-tube transfer programme were subjected to Percoll gradients in order to isolate an enriched population of GL cells. GL cells exhibited specific LPS-binding protein, detected by autoradiography of the cellular lysate on SDS-PAGE after the cells were specifically labelled with a radioiodinated, photoactivable and reducible LPS derivative. LPS binding to the cells was also detected by the appearance of immunofluorescence associated with the cellular membrane when incubated with a fluorescent conjugated LPS receptor antibody. Ninety-four per cent of the cells exhibiting immunofluorescent LPS-binding protein were also positive for the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase, as detected by cytochemistry. In order to detect a response to LPS, the enriched population of GL cells were cultured in vitro in the presence or absence of LPS; after 16 h of culture, tumour necrosis factor-alpha (TNF) mRNA was detected by reverse transcription-polymerase chain reaction and Southern blot analysis of the amplified cDNA. The expression of TNF mRNA was enhanced when the cells were cultured in the presence of LPS, which also significantly enhanced TNF secretion into the media during the 16-h period. These results reveal that GL cells exhibit LPS-binding protein and thus increased TNF secretion occurs in response to LPS in follicular aspirate cells. The source of ovarian TNF may be leukocytes, macrophages and/or GL cells.

Enhanced conditions for DNA fingerprinting with biotinylated M13 bacteriophage.

Deoxyribonucleic acid (DNA) fingerprints are Southern blots which have a pattern resembling bar codes. The pattern is created by DNA probes that bind to variable-length repeated sequences of human genomic DNA digested with restriction endonucleases. To improve DNA fingerprints obtained with biotin-labeled M13mp8 replicative form (RF) bacteriophage as the gene probe, the conditions for hybridization and the subsequent washing steps of the filter were refined. Experiments were conducted varying the electrophoresis time, blotting membranes, hybridization solution, and posthybridization washes. The simplicity, sensitivity, and reliability of this nonistopic technique make possible its application for identification of individuals within a species, for parentage testing, and for monitoring bone marrow transplantation.

Use of silica gel polymer for DNA extraction with organic solvents.

Phenol and chloroform are the standard solvents used for DNA extraction. These solvents aid in the removal of protein and lipid from crude or partially purified cell extracts. Although the procedure is well established, the solvents are noxious, caustic, and unpleasant. We describe in this paper the use of a special blood collection tube to isolate the offensive organic solvents. With the use of silica gel polymer containing tubes, phenol, phenol:chloroform, or chloroform can be separated from the DNA containing aqueous phase in a rapid and safe manner. The method permits higher yields of DNA since the DNA is poured from the tube rather than aspirated with pipet.

Chromosomal assignment of 46 brain cDNAs.

Expressed sequence tags (ESTs) have been obtained from several hundred brain cDNAs as an initial effort to characterize expressed brain genes. These ESTs will become tools for human genome mapping and they will also provide candidate causative genes for inherited disorders affecting the central nervous system. We have developed a procedure for the rapid chromosomal assignment of these ESTs: cDNA sequences are first analyzed by a computer program to determine regions likely not to be interrupted by introns in the genomic DNA. A pair of oligonucleotide primers is then designed to amplify this region by the polymerase chain reaction using DNA template from human-rodent somatic cell hybrid chromosomal panels. The chromosomal assignment of the cDNA is determined by studying the segregation of the amplified products in these panels. In this paper we describe the mapping of 46 brain ESTs, as well as observations on the amplification of rodent sequences.