Characterization of the human neurocan gene, CSPG3.

Neurocan is a chondroitin sulfate proteoglycan thought to be involved in the modulation of cell adhesion and migration. Its sequence has been determined previously in rat and mouse (Rauch et al., 1992. Cloning and primary structure of neurocan, a developmentally regulated, aggregating, chondroitin sulfate proteoglycan of the brain. J. Biol. Chem. 267, 19536-19547; Rauch et al., 1995. Structure and chromosomal location of the mouse neurocan gene. Genomics 28, 405-410). We describe here the complete coding sequence of the human neurocan mRNA, known as CSPG3, as well as mapping data, expression analysis, and genomic structure. A cDNA known as CP-1 was initially sequenced as part of a gene discovery project focused on characterizing chromosome 19-specific cDNAs. Sequence homology searches indicated close homology to the mouse and rat proteoglycan, neurocan (GenBank accession Nos X84727 and M97161). Northern analysis identified a brain-specific transcript of approx. 7.5kb. A longer cDNA clone, GT-5, was obtained, fine-mapped to the physical map of chromosome 19 by hybridization to a chromosome-specific cosmid library, and sequenced. Full coding sequence of the mRNA indicates a 3963bp open reading frame corresponding to a 1321 amino acid protein, similar to the protein length found in mouse and rat. The amino acid sequence of human neurocan shows 63% identity with both the mouse and rat sequences. Finally, genomic sequencing of a cosmid containing the complete neurocan gene was performed to determine the genomic structure of the gene, which spans approx. 41kb, and is transcribed in the telomere to centromere orientation.

Reproducible gene expression measurement among multiple laboratories obtained in a blinded study using standardized RT (StaRT)-PCR.

BACKGROUND: A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility. METHODS AND RESULTS: In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured. CONCLUSION: Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.

Chromosomal assignment of 20 cDNAs using flow-sorted spot-blot stamps.

Using a high-speed flow cytometer/sorter, we constructed spot-blot "stamps" measuring 3.5 x 2.0 cm containing 21 separate human chromosome fractions. Through hybridization to these stamps, 20 randomly selected cDNAs were assigned to specific chromosomes. Sequencing and BLAST database screening confirmed the location of one gene (UCHL1) and allowed the assignment of two other previously identified genes (LRP130 and cDNA IB871.)

Assignment of the human lens fiber cell MP19 gene (LIM2) to chromosome 19q13.4, and adjacent to ETFB.

The LIM2 gene may play a major role in lens fiber cell structure or communication, and thus cataractogenesis. A human cDNA encoding the corresponding lens fiber cell intrinsic protein MP19 has been previously isolated and characterized. This cDNA had been mapped to human chromosome 19. We have independently confirmed this assignment and fine mapped it to 19q13.4. The position of the LIM2 gene appears to be within 40 kb of the electron transport flavoprotein gene (ETFB) as a cosmid containing sequences from both genes has been identified.

Cystatin B-deficient mice have increased expression of apoptosis and glial activation genes.

Loss-of-function mutations in the cystatin B (Cstb) gene cause a neurological disorder known as Unverricht-Lundborg disease (EPM1) in human patients. Mice that lack Cstb provide a mammalian model for EPM1 by displaying progressive ataxia and myoclonic seizures. We analyzed RNAs from brains of Cstb-deficient mice by using modified differential display, oligonucleotide microarray hybridization and quantitative reverse transcriptase polymerase chain reaction to examine the molecular consequences of the lack of Cstb. We identified seven genes that have consistently increased transcript levels in neurological tissues from the knockout mice. These genes are cathepsin S, C1q B-chain of complement (C1qB), beta2-microglobulin, glial fibrillary acidic protein (Gfap), apolipoprotein D, fibronectin 1 and metallothionein II, which are expected to be involved in increased proteolysis, apoptosis and glial activation. The molecular changes in Cstb-deficient mice are consistent with the pathology found in the mouse model and may provide clues towards the identification of therapeutic points of intervention for EPM1 patients.

Isolation, sequencing, and mapping of the human homologue of the yeast transcription factor, SPT5.

We isolated the human homologue, SUPT5H, of the yeast transcription factor, SPT5. The human homologue is 1088 aa long compared to 1063 aa for the yeast gene. SUPT5H maps to 19q13, near the ryanodine receptor. Like its family member, SUPT6H, and like yeast SPT5, SUPT5H has a very acidic 5' domain. Like its family member, SUPT6H, but unlike yeast SPT5 or SPT6, SUPT5H has seven MAP kinase sites at its 5' end. In addition, SUPT5H lacks the novel 6-amino-acid repeat (consensus is S-T/A-W-G-G-A/Q) at the C-terminus of yeast SPT5. This argues that while there is functional similarity between SPT5 and SUPT5H, the molecules differ in the signals to which they respond.