Biomedical subjects

S Korneev

Publications and source records attributed to S Korneev.

4 recordsLinked to original sources

PubMed1999

Cloning, characterisation and expression of the alpha-tubulin genes of the leech, Hirudo medicinalis.

We have isolated two alpha-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two alpha-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo alpha-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech alpha-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod alpha-tubulins. The protein sequences of the leech alpha-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.

Amino Acid Sequence↗

PubMed1996

cDNA libraries from identified neurons.

One approach to studying the changes in gene expression which underlie differentiation is to construct cDNA libraries from different tissues or at different stages of development. However, generating representative cDNA libraries from heterogeneous tissues such as the nervous system is often a real problem. Here, we describe a reproducible method for the construction of large and complex cDNA libraries from a few leech Retzius or P neurons (equivalent to about 50 pg of mRNA) using polymerase chain reaction-based technology. The libraries contain about 10(6) independent recombinants and are remarkably free from contaminating rRNA or polymerase chain reaction artefacts. Sequence analysis of randomly picked clones shows that the libraries contain a high proportion (more than 90%) of cDNAs larger than 500 b.p. As expected, many of the clones are novel, but two (alpha-tubulin and cyclophilin-A) have been extensively characterized in other species. To our knowledge, this is the first report of a cDNA library from identified neurons.

Amino Acid Isomerases↗

PubMed1994

cDNA libraries from a few neural cells.

One of the most powerful approaches to the molecular analysis of differential gene expression is to construct cDNA libraries corresponding to different tissues or developmental stages, and then to enrich for genes expressed in a particular tissue or at a particular time by subtractive hybridisation. Our aim is to reduce the complexity of neuronal cDNA libraries by generating libraries from the mRNA of a single cell. The system chosen is the Retzius cell of the leech, a large neurone which can be unambiguously dissected out. A cDNA library was generated from one leech ganglion (containing about 400 neurons) by anchor 1-oligo dT priming, the addition of dG tails, second strand synthesis primed by an anchor 2-oligo dC primer, followed by PCR from the two anchor regions. XBaI and EcoRI sites were included in the respective anchor primers, between the dT or dC run and the PCR primer sequence, allowing high-efficiency directional cloning. Eight clones picked and sequenced at random gave five with some homology to a known protein and three novel genes. The average insert size in the library was 600 bp, 0.2% of the clones hybridised to repetitive DNA, and 20/30,000 clones gave signals with the Drosophila actin gene. This approach has now been extended to a few pooled Retzius cells.

Animals↗

PubMedDate not supplied

A subtractive cDNA library from an identified regenerating neuron is enriched in sequences up-regulated during nerve regeneration.

We have constructed a subtractive cDNA library from regenerating Retzius cells of the leech, Hirudo medicinalis. It is highly enriched in sequences up-regulated during nerve regeneration. Sequence analysis of selected recombinants has identified both novel sequences and sequences homologous to molecules characterised in other species. Homologies include alpha-tubulin, a calmodulin-like protein, CAAT/enhancer-binding protein (C/EBP), protein 4.1 and synapsin. These types of proteins are exactly those predicted to be associated with axonal growth and their identification confirms the quality of the library. Most interesting, however, is the isolation of 5 previously uncharacterised cDNAs which appear to be up-regulated during regeneration. Their analysis is likely to provide new information on the molecular mechanisms of neuronal regeneration.

Amino Acid Sequence↗