Mutations in exon 17B of cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia.

Pseudoachondroplasia (PSACH) is a well characterized dwarfing condition mapping to chromosome 19p12-13.1. Cartilage oligomeric matrix protein (COMP), a cartilage specific protein, maps to the same location within a contig that spans the PSACH locus. Using single strand conformation polymorphism (SSCP) analysis and nucleotide sequencing we have identified COMP mutations in eight familial and isolated PSACH cases. All mutations involve either a single base-pair change or a three base-pair deletion in exon 17B. Six mutations delete or change a well conserved aspartic acid residue within the calcium-binding type 3 repeats. These results demonstrate that mutations in the COMP gene cause pseudochondroplasia.

Homonyms, synonyms and mutations of the sequence/structure vocabulary.

Calladine (1982) proposed that steric repulsion between adjacent purines on opposite helix backbones accounts for the local variation seen in four helical parameters. By defining simple sum functions based on Calladine 's proposal, Dickerson (1983) has taken what he terms "the first step in establishing a sequence/structure vocabulary". In this letter we analyze the implications of the Calladine - Dickerson model with regard to " homonyms ", "synonyms" and mutations. Specifically, we (1) show that because of the number of adjacent helical positions affected by one transversion, two purine-pyrimidine sequences may be similar at the sequence level yet be very different structurally ( homonyms ); (2) list all sequences which, though different at the sequence level, share adjacent structural parameter values (synonyms); and (3) use two simple statistical measures to show that transversion mutations occurring between a purine and a pyrimidine (5'----3' on the same strand) are in general less disruptive of local helical structure than transversions occurring between a pyrimidine and purine. On the assumption that they are not inconsistent with experimental findings, we discuss the significance of these implications.

Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis. A step towards mapping and sequencing of the genome.

Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector. Thirdly, two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.

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.

Structure, sequence and location of the UQCRFS1 gene for the human Rieske Fe-S protein.

We have identified and studied the chromosomal location of the human Rieske Fe-S protein-encoding gene UQCRFS1. Mapping by hybridization to a panel of monochromosomal hybrid cell lines indicated that a UQCRFS1 partial cDNA was derived from either chromosome 19 or 22. By screening a human chromosome 19 specific genomic cosmid library with a probe from this cDNA sequence, we identified a corresponding cosmid. Portions of this cosmid were sequenced directly. The exon, exon:intron junction and flanking sequences verified that this cosmid contains the genomic locus. Fluorescent in situ hybridization (FISH) was performed to localize this cosmid to chromosome band 19q12.

Periodic structurally similar oligomers are found on one side of the axes of symmetry in the lac, trp, and gal operators.

Three well-defined E. coli operator regions were examined for recurring conformational deviation from a regular B-DNA helix. All three, the lac, trp, and gal, show repeats of the same set of neighboring helical twist angles. These angles recur with a periodicity equal to the helix periodicity on one side of the operator's axes of symmetry. The probability that their occurrence is random was found to be extremely small. Therefore, we propose that in addition to specific bases, repeating twist angles patterns are likely to be among the local parameters involved in repressor-operator recognition.

Structural features are as important as sequence homologies in Drosophila heat shock gene upstream regions.

Pelham has shown that the Drosophila hsp 70 gene is not transcribed under heat shock conditions unless a given upstream region is present. Davidson et al. have recently compiled a list of sequences homologous to this region in other Drosophila heat shock genes. They proposed that a set of unlinked genes, such as the heat shock genes, could be coordinately induced through an interaction in cis with a common regulatory molecule. That this interaction involves structural elements is suggested by the fact that these upstream regions share inverted repeats as well as areas of Z-DNA potential. Furthermore, using the Calladine-Dickerson rules for local helical parameters, we show that these regions share structural homology. This is significant because the presence of regions homologous to a derived consensus sequence does not necessarily imply structural similarity. Therefore, we suggest that these structural features are at least as important as the sequence homologies in enabling the heat shock response.

Gene database for the fission yeast Schizosaccharomyces pombe.

As an aid to the fission yeast genome project, we describe a database for Schizosaccharomyces pombe consisting of both genetic and physical information. As presented, it is therefore both an updated gene list of all the nuclear genes of the fission yeast, and provides an estimate of the physical distance between two mapped genes. Additionally, a field indicates whether the sequence of the gene is available. Currently, sequence information is available for 135 of the 501 known genes.

C mu-containing transcripts initiate heterogeneously within the IgH enhancer region and contain a novel 5'-nontranslatable exon.

Transcriptional competence of the immunoglobulin heavy-chain locus (IgH) is established at an early stage of lymphoid cell development, leading to the appearance of RNA components, previously called C mu RNA1 or sterile-mu RNA2, which contain constant-region sequences but lack variable-region sequences. These components are of two types: those which initiate in the D region of alleles that have undergone DJH (diversity-joining region) rearrangement (D mu transcripts) and those which initiate within the JH-C mu intron (hereafter termed I mu transcripts). In pre-B and early B cells, D mu and I mu transcripts are nearly as abundant as the messenger RNA encoding mu heavy chain. The D mu transcripts are spliced into RNAs containing D, JH and C mu sequences, and in some, but not all, cases these RNAs are translated into D mu proteins. To establish whether the I mu transcripts have any translational potential and to elucidate the structure of their promoter region, we have determined their transcription initiation sites and their mode of splicing. As reported here, by using sequence analysis of cloned I mu complementary DNAs, primer extension and S1 nuclease mapping, we have found that these transcripts have remarkable 5' heterogeneity: there are more than five distinct start sites spanning a region of 44 nucleotides that is located downstream of an octanucleotide found in all variable-region promoters. Such imprecise initiation may result from the lack of a well-defined TATAA motif and the unusual proximity of the octanucleotide to the enhancer region. Approximately 700 nucleotides downstream from these initiation sites, a cryptic splice site is used to create a nontranslatable exon ('nontron') which is joined to the C mu 1 domain. The properties of the nontron may be important for the mechanism of allelic exclusion.

Eukaryotic oligomer frequencies are correlated with certain DNA helical parameters.

Nucleotide sequences were converted into purine (R)-pyrimidine (Y) series and divided into several groups, embracing higher and lower organisms. The frequencies of R-Y doublets, triplets and quartets in each were calculated. Whereas eukaryotes uniformly show RR + YY greater than RY + YR, in bacteria and phage no such relationship is observed. The triplet and quartet patterns in higher organisms differ from those seen in prokaryotes. In the higher organisms a correlation is observed between the frequencies of triplets and quartets and some DNA structural parameters. Specifically, the most frequent triplets are those with minimal torsion angle deviations from a regular B-DNA. The most frequent quartets are those with minimal roll angle deviations. No such correlations are observed in prokaryotes. We therefore propose that in eukaryotic DNA, tight, smooth packaging imposes sequence constraints.

CpG frequency in large DNA segments.

The relationship between the deficiency of CpG dinucleotides and the coding-noncoding segments of DNA has been examined. Analysis of five human alpha-like globin DNA sequences and five human beta-like globin DNA sequences reveal that there is no apparent difference between protein coding and non-coding portions of DNA. Rather CpG deficiency appears to be a property of long contiguous segments of DNA consisting of several genes and their intergenic regions. Thus we propose that CpG deficiency is not involved with translation or transcription but rather is related to chromosomal constraints.

The temporal order of appearance of transcripts from unrearranged and rearranged Ig genes in murine fetal liver.

The developmental time course of RNA transcribed from unrearranged (germ-line) and rearranged Ig genes in murine fetal liver was determined by a quantitative Northern blot analysis. Sterile Cmu transcripts and germ-line VH transcripts are detectable as early as day 14, whereas significant amounts of rearranged VDJCmu H chain transcripts do not appear until days 16 to 17. The sterile Cmu transcripts continuously increase in abundance throughout fetal development, in contrast to the germ-line VH transcripts, which decrease abruptly after day 16. Transcripts of germ-line and rearranged CK genes are detectable on day 17, and continue to increase in abundance on day 18. Transcripts from a pre-B cell specific gene, lambda 5, first appear on day 15, and reach maximum abundance on day 17. The order of these events is consistent with the known order of gene rearrangements and with the idea that transcriptional activation of germ-line loci is a prerequisite for Ig gene rearrangement. The lag between the onsets of germ-line Cmu and VH transcription and the appearance of VDJCmu transcripts suggests that additional developmentally regulated events may be necessary to achieve efficient expression of completely rearranged H chain genes.

The identification of exons from the MED/PSACH region of human chromosome 19.

We have used exon amplification to identify putative transcribed sequences from an 823-kb contig consisting of 28 cosmids that form a minimum tiling path from the interval 19p12-p13.1. This region contains the genes responsible for multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). We have trapped 66 exons (an average of 2.4 exons per cosmid) from pools of 2 or 3 cosmids. The majority of exons (51.5%) show only weak similarity or no similarity (36.3%) to sequences in current databases. Six of 8 exons examined from these groups, however, show cross-species sequence conservation, indicating that many of them probably represent authentic exons. Eight exons show identity or significant similarity to ESTs or known genes, including the human TNF receptor 3 '-flanking region gene, human epoxide hydrolase (EPHX), human growth/differentiation factor (GOF-1), human myocyte-specific enhancer factor 2, the rat neurocan gene, and the human cartilage oligomeric matrix protein gene (COMP). Mutations in this latter gene have recently been shown to be responsible for MED and PSACH.

Transcription of unrearranged antigen receptor genes in scid mice.

The scid mouse mutant is severely deficient in lymphocytes; cells of the B or T lymphocyte lineage cannot be detected by either serological or functional assays. However, as shown here, germ-line transcripts of B cell immunoglobulin (Ig) constant and variable region genes and of T cell receptor (TCR) genes are detectable in lymphopoietic tissues of scid mice, as well as B and T lineage-specific lambda 5 and T3 delta transcripts. We conclude that B and T lineage-committed cells do arise in scid mice and that their Ig and TCR genes are accessible to enzymes involved in their recombination. This suggests that scid impairs lymphopoiesis at the stage at which antigen receptor genes normally undergo rearrangement.

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.)

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.

Hybridization fingerprinting of high-density cDNA-library arrays with cDNA pools derived from whole tissues.

As part of an integrated mapping and sequencing analysis of genomes, we have developed an approach allowing the characterization of large numbers of cDNA library clones with a minimal number of experiments. Three basic elements used in the analysis of cDNA libraries are responsible for the high efficiency of this new approach: (1) high-density library arrays allowing thousands of clones to be screened simultaneously; (2) hybridization fingerprinting techniques to identify clones abundantly expressed in specific tissues (by hybridizations with labeled tissue cDNA pools) and to avoid the repeated selection of identical clones and of clones containing noncoding inserts; and (3) a computerized system for the evaluation of hybridization data. To demonstrate the feasibility of this approach, we hybridized high-density cDNA library arrays of human fetal brain and embryonal Drosophila with radiolabeled cDNA pools derived from whole mouse tissues. Fingerprints of the library arrays were generated, localizing clones containing cDNA sequences from mRNAs expressed at middle to high abundance (> 0.1-0.15%) in the respective tissue. Partial sequencing data from a number of clones abundantly expressed in several tissues were generated to demonstrate the value of the approach, especially for the selection of cDNA clones for the analyses of genomes based on expressed sequence tagged sites. Data obtained by the technique described will ultimately be correlated with additional transcriptional and sequence information for the same library clones and with genomic mapping information in a relational database.