Assignment of genes encoding a unique cytokine (IL12) composed of two unrelated subunits to chromosomes 3 and 5.

IL12 (formerly NKSF or CLMF) is a unique cytokine composed of two unrelated disulfide-linked subunits. The larger 40-kDa subunit (p40) is a member of the cytokine receptor family, and the smaller 35-kDa subunit (p35) is related to IL6 and GCSF. The chromosomal localization of these two subunits was determined by PCR analysis of DNA from rodent-human hybrids. More refined mapping was obtained by PCR analysis of hybrids containing translocation chromosomes and for p40, by analysis of radiation hybrids. The subunits map to different chromosomes: p40 (IL12B) to 5q31-q33 and p35 (IL12A) to 3p12-3q13.2.     

Human dopamine transporter gene (DAT1) maps to chromosome 5p15.3 and displays a VNTR.

The human dopamine transporter (DAT1) gene is localized to chromosome 5p15.3 by in situ hybridization and PCR amplification of rodent somatic cell hybrid DNA. Analysis of a 40-bp repeat in the 3' untranslated region of the message revealed variable numbers of the repeat ranging from 3 to 11 copies. These results will aid in the investigation of a role for this gene in genetic disorders of the dopaminergic system in humans.     

Regional localization of DNA sequences on chromosome 21 using somatic cell hybrids.

We have used a panel of Chinese hamster X human somatic cell hybrids, each containing various portions of chromosome 21 as the only detectable human chromosome component, for regional mapping of cloned, chromosome 21-derived DNA sequences. Thirty unique and very low-repeat sequences were mapped to the short arm and three sections of the long arm. Three unique sequences map to the proximal part of the terminal band 21q22.3, and five to the distal part of this band. Some of these may represent parts of gene sequences that may be relevant to the pathogenesis of Down syndrome, as 21q22 is the area required to be present in triplicate for the full clinical picture.     

High performance liquid chromatography and time-of-flight secondary ion mass spectrometry: a new dimension in structural analysis of apolipoproteins

We report the isolation and characterization of an apolipoprotein A-I mutant using a new technique for structural analysis of apolipoproteins based upon the combined techniques of protein isolation by isoelectric focusing in immobilized pH-gradients, reversed-phase HPLC of tryptic peptides, and subsequent molecular weight analysis of isolated peptides by time-of-flight secondary ion mass spectrometry (TOF-SIMS). The particular advantages of the TOF-SIMS procedure in the characterization of proteolytic peptides are the detection limits in the picomole range, the accuracy of molecular weight determination (up to 3000 +/- 1 D), the speed of analysis, and the wide range of applications for involatile biomolecules. The described procedure for the analysis of apolipoproteins requires only 2 ml of serum as starting material. This method can be used to monitor for genetic polymorphisms and posttranslational modifications on a microscale basis. Applying these techniques, we characterized a new apolipoprotein A-I mutant with an amino acid exchange arginine177 by histidine.     

FGFR2 exon IIIa and IIIc mutations in Crouzon, Jackson-Weiss, and Pfeiffer syndromes: evidence for missense changes, insertions, and a deletion due to alternative RNA splicing.

Fibroblast growth factor receptor 2 (FGFR2) mutations have been associated with the craniosynostotic conditions Crouzon, Jackson-Weiss, and Pfeiffer syndromes. Previously, mutations were described in the exons IIIa and IIIc, which form the extracellular, third immunoglobulin-like domain (IgIII) and adjacent linker regions, both of which are normally involved in ligand binding. For all three conditions, mutations were found in exon IIIc. Only in Crouzon syndrome were mutations identified in exon IIIa. In this study, 39 cases with one of these three conditions were screened for exon IIIa or IIIc mutations. Eleven mutations are reported in 17 unrelated cases. Mutations in exon IIIa are identified for not only Crouzon but also Jackson-Weiss and Pfeiffer syndromes. Four mutations in either exon IIIa or exon IIIc reported only in Crouzon syndrome are present also in one of the other two syndromes. Two insertions, one in exon IIIa in a Crouzon syndrome patient and the other in exon IIIc in a Pfeiffer syndrome patient, were observed. The latter mutation has the same alternative RNA splicing effect as a reported synonymous mutation for Crouzon syndrome. A missense mutation was detected in one Pfeiffer syndrome family in which two members had craniosynostosis without limb anomalies. The inter- and intrafamilial variability in expression of FGFR2 mutations suggests that these three syndromes, presumed to be clinically distinct, are instead representative of a spectrum of related craniosynostotic and digital disorders.     

Human gamma X satellite DNA: an X chromosome specific centromeric DNA sequence

The cosmid clone, CX16-2D12, was previously localized to the centromeric region of the human X chromosome and shown to lack human X-specific satellite DNA. A 1.2 kb EcoRI fragment was subcloned from the CX16-2D12 cosmid and was named 2D12/E2. DNA sequencing revealed that this 1,205 bp fragment consisted of approximately five tandemly repeated DNA monomers of 220 bp. DNA sequence homology between the monomers of 2D12/E2 ranged from 72.8% to 78.6%. Interestingly, DNA sequence analysis of the 2D12/E2 clone displayed a change in monomer unit orientation between nucleotide positions 585–586 from a tail-to-head arrangement to a head-to-tail configuration. This may reflect the existence of at least one inversion within this repetitive DNA array in the centromeric region of the human X chromosome. The DNA consensus sequence derived from a compilation of these 220 bp monomers had approximately 62% DNA sequence similarity to the previously determined 8 satellite DNA consensus sequence. Comparison of the 2D12/E2 and 8 consensus sequences revealed a 20 bp DNA sequence that was well conserved in both DNA consensus sequences. Slot-blot analysis revealed that this repetitive DNA sequence comprises approximately 0.015% of the human genome, similar to that found with 8 satellite DNA. These observations suggest that this satellite DNA clone is derived from a subfamily of satellite DNA and is thus designated X satellite DNA. When genomic DNA from six unrelated males and two unrelated females was cut with SstI or HpaI and separated by pulsed-field gel electrophoresis, no restriction fragment length polymorphisms were observed for either X (2D12/E2) or 8 (50E4) probes. Fluorescence in situ hybridization localized the 2D12/E2 clone to the lateral sides of the primary constriction specifically on the human X chromosome.     

A centromere-based genetic map of the short arm of human chromosome 6

A genetic map of the short arm of chromosomes 6 (6p) has been constructed with 20 genetic markers that define 16 loci, including a locus at the centromere. The 40 CEPH families and, for 4 loci, 13 additional Utah families were genotyped. All 16 loci form a single linkage group extending from near the telomeric region to the centromere, covering 159 cM (Haldane) on the female map and 94 cM on the male map. Sex differences in recombination frequencies are noted for the 6p map, with an excess occurring in males at the distal end. The genetic order of loci is consistent with their physical localization on 6p. Proximal to the three most distal loci on the map, markers are especially dense, providing an extended region on 6p useful for localizing genes of interest.     

Chromosomal deletion 4p15.32----p14 in a Treacher Collins syndrome patient: exclusion of the disease locus from and mapping of anonymous DNA sequences to this region.

Treacher Collins syndrome is an autosomal dominant condition of bilateral craniofacial abnormalities of structures derived from the first and second branchial arches. A patient with severe manifestations of Treacher Collins syndrome and a de novo chromosomal deletion in region 4p15.32----p14 was identified. Anonymous DNA sequences of loci D4S18, D4S19, D4S20, D4S22, and D4S23 were mapped to the deleted region. DNA probes previously mapped to loci on chromosome 4p (D4S10, D4S15, D4S16, D4S26, D4S35, D4S95, D4S144, RAF1P1, QDPR, and HOX7) were not deleted in this patient. Linkage analysis between the D4S18, D4S23, and QDPR loci and Treacher Collins syndrome in eight families excluded the Treacher Collins syndrome locus from the region of the deletion.     

Cellulose-bound Peptide Arrays: Preparation and Applications

Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of cellular phenomenon is the purinosome, a newly discovered multi-enzyme metabolic assembly that includes all of the enzymes within the de novo purine biosynthetic pathway. Our understanding of the components and regulation of purinosomes has significantly grown in recent years. This paper reviews the purine de novo biosynthesis pathway and its regulation, and presents the evidence supporting the purinosome assembly and disassembly processes under the control of G-protein-coupled receptor (GPCR) signaling. This paper also discusses the implications of purinosome and GPCR regulation in drug discovery.