Integrated mapping analysis of the Werner syndrome region of chromosome 8

The Werner syndrome locus (WRN) is located at 8p11-p12. To facilitate eventual cloning of the WRN gene, a 10,000-rad radiation-reduced hybrid (RH) cell panel was generated to map genetic markers, sequence-tagged sites (STSs), and genes in this region. A hamster cell line carrying an intact human chromosome 8 was fused with another hamster cell line. Two sets of hybrid cell panels from 2 separate fusions were generated; each panel consisted of 50 independent clones; 33 and 34 cell lines from the 2 fusions retained human chromsome material as determined by inter-Alu PCR. The combined panel was genotyped for 52 markers spanning the entire chromosome, including 10 genes, 29 anonymous polymorphic loci, and 13 STSs. Seventeen of these markers have not been previously described. Markers near the centromere were retained at a higher frequency than more distal markers. Fluorescence in situ hybridization was also used to localize and order a subset of the markers. A RH map of the WRN region was constructed using a maximum likelihood method, giving the following most likely order: D8S131-D8S339 (GSR)-D8S124-D8S278-D8S259-(D8S71)-D8S283- D8S87-D8S105-D8S135 (FGFR1)-D8S135PB-D8S255-ANK1. A genetic map of 15 short tandem repeat polymorphic loci in the WRN region was also constructed. The marker orders from the genetic and RH maps were consistent. In addition, an integrated map of 24 loci in the WRN region was generated using information from both genetic and RH mapping methods. A 1000:1 framework map for 6 loci (LPL-D8S136-D8S137-D8S87-FGFR1-ANK1) was determined by genetic mapping, and the resulting locus order was fixed during analysis of the RH genotype data. The resulting integrated map contained more markers than could confidently be ordered by either genetic or RH mapping alone.     

A transcript map of the newly defined 165 kb Wolf-Hirschhorn syndrome critical region

The nucleotide sequence of the bphB gene of Pseudomonas putida strain OU83 was determined. The bphB gene, which encodes cis-biphenyl dihydrodiol dehydrogenase (BDDH), was composed of 834 base pairs with an ATG initiation codon and a TGA termination codon. It can encode a polypeptide of 28.91 kDa, containing 277 amino acids. Promoter-like and ribosome-binding sequences were identified upstream of the bphB gene. The bphB nucleotide sequence was used to produce His-tagged BDDH, in Escherichia coli. The His-tagged BDDH construction, carrying a single 6 x His tail on the N-terminal portion, was active. The molecular mass of the native enzyme was 128 kDa and on SDS-PAGE analysis the molecular mass was 31 kDa. This enzyme requires NAD+ for its activity and its optimum pH is 8.5. Nucleotide and the deduced amino acid sequence analyses revealed a high degree of homology between the bphB gene from Pseudomonas putida OU83 and the bphB genes from P. cepacia LB400 and P. pseudoalcaligenes KF707.     

Refined linkage map of chromosome 5 in the region of the spinal muscular atrophy gene

The genetic map in the region of human chromosome 5 that harbors the gene for autosomal recessive forms of spinal muscular atrophy (SMA) has been refined by a multilocus linkage study in 50 SMA-segregating families. Among six markers spanning 8 cM for combined sexes, four were shown to be tightly linked to the SMA locus. Multipoint linkage analysis was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for the SMA locus is between blocks AFM114ye7 (D5S465)/EF5.15 (D5S125) and MAP-1B/JK53 (D5S112) at a sex-combined genetic distance of 2.4 and 1.7 cM, respectively. Thus the SMA gene lies in the 4-cM region between these two blocks. This information is of primary importance for designing strategies for isolating the SMA gene.     

Localization of the gene for Wieacker-Wolff syndrome in the pericentromeric region of the X chromosome

The Wieacker-Wolff syndrome (WWS, MIM* 314580), first described clinically in 1985, is an X-linked recessive disorder. In earlier studies, linkage between the WWS gene and DXYS1 at Xq21.2 and DXS1 at Xq11 as well as AR at Xq12 was reported. Here we report on a linkage analysis using highly polymorphic, short terminal repeat markers located in the segment from Xp21 to Xq24. No recombination between the WWS locus and ALAS2 or with AR (z = 4.890 at theta = 0.0) was found. Therefore, the WWS locus was assigned to a segment of approximately 8 cM between PFC (Xp11.3-Xp 11.23) and DXS339 (Xq11.2-Xq13).     

A gene for FG syndrome maps in the Xq12-q21.31 region

AIMS: An increased risk of adenocarcinoma of the oesophagus has been demonstrated in patients with long segments of Barrett's mucosa. The risk of cancer associated with short segments of metaplasia of the oesophagogastric junction is not known. METHODS AND RESULTS: We report a case of early adenocarcinoma of the oesophagus arising on short tongues of Barrett's mucosa associated with an oesophageal cyst. The patient was a 68-year-old man with no previous clinical history of gastro-oesophageal reflux disease. The fortuitous discovery of an oesophageal cyst lead to the diagnosis of short tongues of Barrett's mucosa with high-grade dysplasia. On pathological examination of the resected specimen, an early adenocarcinoma had developed in Barrett's mucosa, localized just above the oesophageal cyst. CONCLUSIONS: As oesophageal cysts can cause symptoms suggestive of reflux, we hypothesize that this association may not be fortuitous.     

Solid-phase combinatorial chemistry and novel tagging methods for identifying leads

Encoded combinatorial chemical synthesis on solid phase is a new paradigm in organic chemistry that provides chemists with powers similar to those enjoyed by molecular biologists. Encoded chemical libraries will have a profound impact on all endeavors that seek to identify molecules with optimized properties and to understand the factors governing molecular interactions. In particular, the discovery and optimization of new therapeutic and diagnostic drug molecules, traditionally a slow manual process, will be greatly accelerated by this technology.     

Complete physical map of the common deletion region in Williams syndrome and identification and characterization of three novel genes

Williams syndrome (WS) is a contiguous gene deletion disorder caused by haploinsufficiency of genes at 7q11.23. We have shown that hemizygosity of elastin is responsible for one feature of WS, supravalvular aortic stenosis (SVAS). We have also implicated LIM-kinase 1 hemizygosity as a contributing factor to impaired visual-spatial constructive cognition in WS. However, the common WS deletion region has not been completely characterized, and genes for additional features of WS, including mental retardation, infantile hypercalcemia, and unique personality profile, are yet to be discovered. Here, we present a physical map encompassing 1.5 Mb DNA that is commonly deleted in individuals with WS. Fluorescence in situ hybridization analysis of 200 WS individuals shows that WS individuals have the consistent deletion interval. In addition, we identify three novel genes from the common deletion region: WS-betaTRP, WS-bHLH, and BCL7B. WS-betaTRP has four putative beta-transducin (WD40) repeats, and WS-bHLH is a novel basic helix-loop-helix leucine zipper (bHLHZip) gene. BCL7B belongs to a novel family of highly conserved genes. We describe the expression profile and genomic structure for each of these genes. Hemizygous deletion of one or more of these genes may contribute to developmental defects in WS.     

Comparison of gene probe and conventional methods for the differentiation of ovine footrot isolates of Dichelobacter nodosus

In a collaborative study that involved four Australian veterinary diagnostic laboratories a gene probe test based on the recombinant plasmids pJIR318, pJIR314B, and pJIR313, which contain genomic vap or vrl regions, was compared with conventional tests used for the differential diagnosis of ovine footrot. A total of 771 clinical dichelobacter nodosus isolates were tested and designated as belonging to one of several gene probe categories. The results showed that 87% of the virulent isolates belonged to gene probe category 1, compared to only 6% of the benign isolates. It was concluded that there was good correlation between the gene probe test and the virulence designation of these isolates as well as the results of elastase, gelatin-gel and protease isoenzyme tests. Furthermore, the gene probe test was converted to a polymerase chain reaction (PCR)-based test. It is suggested that diagnostic laboratories consider carrying out both this PCR test and tests based on the extracellular proteases of D. nodosus.     

Bacterial contig map of the 21q11 region associated with Alzheimer's disease and abnormal myelopoiesis in Down syndrome

We present a high-resolution bacterial contig map of 3.4 Mb of genomic DNA in human chromosome 21q11-q21, encompassing the region of elevated disomic homozygosity in Down Syndrome-associated abnormal myelopoiesis and leukemia, as well as the markers, which has shown a strong association with Alzheimer's Disease that has never been explained. The map contains 89 overlapping PACs, BACs, or cosmids in three contigs (850, 850, and 1500 kb) with two gaps (one of 140-210 kb and the second <5 kb). To date, eight transcribed sequences derived by cDNA selection, exon trapping, and/or global EST sequencing have been positioned onto the map, and the only two genes so far mapped to this cytogenetic region, STCH and RIP140 have been precisely localized. This work converts a further 10% of chromosome 21q into a high-resolution bacterial contig map, which will be the physical basis for the long-range sequencing of this region. The map will also enable positional derivation of new transcribed sequences, as well as new polymorphic probes, that will help in elucidation of the role the genes in this region may play in abnormal myelopoiesis and leukemia associated with trisomy 21 and Alzheimer's Disease.     

Comparison of four RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus by RT-PCR

We investigated the effect of repeated cold stress (RCS) on the capsaicin-evoked release of glutamate from the primary afferent fibers of the rat, and compared this with the effect of inoculation of complete Freund's adjuvant (adjuvant inoculation). The release of glutamate was measured using a fluorometric on-line continuous monitoring system in which the immobilized glutamate dehydrogenase column was connected to an in vitro superfusion system. In the presence of 0.3 microM tetrodotoxin, the application of 1 microM capsaicin to spinal dorsal horn slices evoked glutamate release (18.6 +/- 1.2 pmol mg(-1) protein, n = 11). In rats subjected to RCS (RCS rats), the release of glutamate evoked by 1 microM capsaicin was markedly increased to 272% (n = 6, P < 0.05) of the value for the control group, although the basal release was not significantly altered (n = 6, P > 0.05). Adjuvant inoculation produced a significant increase in the basal and capsaicin (1 microM) evoked release of glutamate to 141 and 344% (n = 6, P < 0.05) of the value for the control group, respectively. The present results suggest that the facilitated release of glutamate from capsaicin-sensitive primary afferent terminals in the spinal dorsal horn is, at least in part, involved in the hyperalgesia of RCS rats as well as the complete Freund's adjuvant-induced hyperalgesia.     

A high-resolution PAC and BAC map of the SCA2 region

DNA sequence changes induced in the vermilion gene of Drosophila following in vivo treatment of postmeiotic male germ cells with vinyl carbamate (VCA), an etheno-adduct-forming carcinogen, are primarily deletions. With VCA, 65% (13/20) of the vermilion mutants isolated from crosses of NER+ (nucleotide excision repair) males with NER+ females and 40% (6/15) obtained from matings with NER- females were intra- or multi-locus deletions. Due to the insufficiently low mutagenic activity in NER+ genotypes of vinyl bromide (VB), another epsilon-adduct-forming carcinogen, vermilion mutants could only be isolated from crosses of VB-treated males with NER- females. Of 14 vermilion mutants induced by VB, three carried large deletions. Twenty-two of 23 base substitutions derived from either VCA or VB experiments fell into one of the four categories expected from epsilon-adducts: three vermilion mutants had GC-->AT transitions, five had AT-->GC transitions, 7 carried GC-->TA transversions, and 7 were AT-->TA transversions. In view of the similarities in the response of mouse and Drosophila germ lines to several classes of alkylating agents, a high incidence of deletions is predicted to occur as well in postmeiotic germ cells of mice exposed to these types of agents.     

Comparative mapping of the DiGeorge syndrome region in mouse shows inconsistent gene order and differential degree of gene conservation

We have constructed a comparative map in mouse of the critical region of human 22q11 deleted in DiGeorge (DGS) and Velocardiofacial (VCFS) syndromes. The map includes 11 genes potentially haploinsufficient in these deletion syndromes. We have localized all the conserved genes to mouse Chromosome (Chr) 16, bands B1-B3. The determination of gene order shows the presence of two regions (distal and proximal), containing two groups of conserved genes. The gene order in the two regions is not completely conserved; only in the proximal group is the gene order identical to human. In the distal group the gene order is inverted. These two regions are separated by a DNA segment containing at least one gene which, in the human DGS region, is the most proximal of the known deleted genes. In addition, the gene order within the distal group of genes is inverted relative to the human gene order. Furthermore, a clathrin heavy chain-like gene was not found in the mouse genome by DNA hybridization, indicating that there is an inconsistent level of gene conservation in the region. These and other independent data obtained in our laboratory clearly show a complex evolutionary history of the DGS-VCFS region. Our data provide a framework for the development of a mouse model for the 22q11 deletion with chromosome engineering technologies.     

Comparison of dose standard units for drug utilisation studies

Primary malignant melanoma of the esophagus is rare. We identified one patient over a period of 15 years. This patient was a 80 years-old caucasian man. No association was found with tobacco or ethanol use, nor was there a personal or family history of malignant melanoma. Symptoms were related to obstruction. This tumor was polypoid in its upper part and ulcero-infiltrant in its lower part. Histologically the melanoma had epithelioid spindle cells. The neoplasm was immunoreactive for S-100 protein and non reactive with anti-cytokeratins. This patient was treated by Garlock type, esophagectomy with excision of 13 cm of esophagus and 2 cm of stomach. The survival was of only 3 days, because he developed acute respiratory and cardiac disease syndrome and died.