[Accidentally discovered vascular abnormality].
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Adult↗
Biomedical subjects
J Rommens
Publications and source records attributed to J Rommens.
Molecular genetics of cystic fibrosis.
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Animals↗
Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
The gene responsible for cystic fibrosis, the most common severe autosomal recessive disorder, is located on the long arm of human chromosome 7, region q31-q32. The gene has recently been identified and shown to be approximately 250 kb in size. To understand the structure and to provide the basis for a systematic analysis of the disease-causing mutations in the gene, genomic DNA clones spanning different regions of the previously reported cDNA were isolated and used to determine the coding regions and sequences of intron/exon boundaries. A total of 22,708 bp of sequence, accounting for approximately 10% of the entire gene, was obtained. Alignment of the genomic DNA sequence with the cDNA sequence showed perfect colinearity between the two and a total of 27 exons, each flanked by consensus splice signals. A number of repetitive elements, including the Alu and Kpn families and simple repeats, such as (GT)17, (GATT)7, and (TA)14, were detected in close vicinity of some of the intron/exon boundaries. At least three of the simple repeats were found to be polymorphic in the population. Although an internal amino acid sequence homology could be detected between the two halves of the predicted polypeptide, especially in the regions of the two putative nucleotide-binding folds (NBF1 and NBF2), the lack of alignment of the nucleotide sequence as well as the different positions of the exon/intron boundaries does not seem to support the hypothesis of a recent gene duplication event. To facilitate detection of mutations by direct sequence analysis of genomic DNA, 28 sets of oligonucleotide primers were designed and tested for their ability to amplify individual exons and the immediately flanking sequences in the introns.
Amino Acid Sequence↗
A cluster of highly polymorphic dinucleotide repeats in intron 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
A cluster of highly polymorphic dinucleotide repeats has been detected in intron 17b of the CFTR gene, 200 bp downstream from the preceding exon. At least 24 alleles, with sizes ranging from 7 to 56 units of a TA repeat, have been identified in a panel of 92 unrelated carriers of cystic fibrosis (CF). The common ones are those with 7, 30, and 31 dinucleotide units, with frequencies of .22, .19, and .12, respectively, among the non-CF chromosomes. Mendelian, codominant segregation of the alleles has been demonstrated in family studies, as expected. A less polymorphic dinucleotide (CA repeat) cluster has also been detected in a region 167 bp downstream from the TA repeat. The length of the CA repeat cluster varies from 11 to 17 dinucleotide units, and it appears to have an inverse relationship to that of the TA repeats. These dinucleotide repeats should be useful in genetic linkage studies, in counseling for CF families with unknown mutations, and in tracing the origins of the various mutant CF alleles.
Adenine Nucleotides↗
The relation between genotype and phenotype in cystic fibrosis--analysis of the most common mutation (delta F508).
BACKGROUND AND METHODS: Both the clinical manifestations of cystic fibrosis and the genotypes of patients are heterogeneous, but the associations between the two are not known. We therefore studied blood samples from 293 patients with cystic fibrosis for the presence of the most common disease-causing mutation (delta F508) on chromosome 7 and compared the results with the clinical manifestations of the disease. RESULTS: The prevalence of the delta F508 allele in the cohort was 71 percent; 52 percent of the patients were homozygous for the mutation, 40 percent were heterozygous, and 8 percent had other, undefined mutations. The patients who were homozygous for the mutation had received a diagnosis of cystic fibrosis at an earlier age and had a greater frequency of pancreatic insufficiency; pancreatic insufficiency was present in 99 percent of the homozygous patients, but in 72 percent of the heterozygous patients and only 36 percent of the patients with other genotypes. The patients with pancreatic insufficiency in all three genotype groups had similar clinical characteristics, reflected by an early age at diagnosis, similar sweat chloride values at diagnosis, similar severity of pulmonary disease, and similar percentiles for weight. In contrast, the patients in the heterozygous-genotype and other-genotype groups who did not have pancreatic insufficiency were older and had milder disease. They had lower sweat chloride values at diagnosis, normal nutritional status, and better pulmonary function after adjustment for age. CONCLUSIONS: The variable clinical course in patients with cystic fibrosis can be attributed at least in part to specific genotypes at the locus of the cystic fibrosis gene.
Adolescent↗
Isolation of clones on chromosome 7 that contain recognition sites for rare-cutting enzymes by oligonucleotide hybridization.
Five G/C-containing oligonucleotides that include the recognition sequences of rare-cutting restriction enzymes have been used to isolate almost 100 different genomic segments from chromosome 7 that contain recognition sites for those enzymes. Hybridization and washing at 27 degrees C allow the use of 8-bp radiolabeled oligonucleotides to detect specific G/C-containing sequences in less than 1 ng of cloned DNA. This method was used to isolate 9 positive clones from 138 previously isolated single-copy probes from a flow-sorted chromosome 7 library. The specificity of the method was confirmed by showing that clones that gave positive hybridization signals also contained the corresponding restriction site. The oligonucleotides were also used to analyze approximately 12,000 kb of genomic sequence from a newly constructed chromosome 7 cosmid library that yielded 88 positive cosmids from 350 analyzed. The average distances between binding sites ranged from 200 to 690 kb and was independent of the number of CpG residues present in the oligonucleotide. Confirmation that clones containing restriction sites for these rare-cutting enzymes are located near genes was obtained by hybridization to RNA and cross-species DNA blots.
Animals↗
Rapid nonradioactive detection of the major cystic fibrosis mutation.
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Chromosome Deletion↗
Chemical synthesis and characteristics of a hybrid phage T5-lac promoter.
A new P1O1R9 inducible promoter (where P1 is a promoter sequence analogous to that of the phage T5 early promoter; O1 is lac-operator; and R9 is a ribosome binding site) was synthesized. We studied the efficiency of this promoter in controlling and inducible gene expression using two model genes: human calcitonin tetrameric (hCT[4]) and human interferon alpha 1 (hIFN alpha 1). The synthetic lac-operator O1 was found to repress P1 activity in media free of lac-operon inducers which was derepressed in the presence of IPTG. The levels of expression of both genes (evaluated by quantitating mRNA and protein) in the presence of lac-inducers were close to those obtained with the P1R9 constitutive promoter.
Base Sequence↗
Chemical synthesis of a biologically active gene for human immune interferon-gamma. Prospect for site-specific mutagenesis and structure-function studies.
A 453-base pair DNA duplex consisting of a gene coding for human interferon-gamma and initiation and termination signals plus appropriate restriction enzyme sites for plasmid insertion has been totally synthesized. The synthesis involved preparation of 66 oligodeoxynucleotides by a modified, solid phase phosphite procedure and enzymatic ligation of the oligonucleotides. The gene, when inserted into a previously constructed expression vector, was expressed in Escherichia coli, demonstrating functional activity for the synthetic gene. Several strategically located restriction cleavage sites have been introduced into the sequence. This provides a convenient system for site-specific mutagenesis for structure-function studies.
Amino Acid Sequence↗
High-level expression of a chemically synthesized gene for human interferon-gamma using a prokaryotic expression vector.
A chemically synthesized gene for human interferon-gamma has been cloned into a prokaryotic expression vector under the regulation of a synthetic constitutive transcriptional-translational control unit that contains a strong bacteriophage T5 early promoter and a strong ribosome-binding site. Cells harboring the recombinant plasmid express high levels (4 X 10(9) units per liter of culture) of antiviral activity specific for interferon-gamma. Analysis of total cell lysates on NaDodSO4/polyacrylamide gels revealed a 17,200-dalton protein, expected for the nonglycosylated form of human interferon-gamma, that constitutes greater than 15% of total cell protein.
Amino Acid Sequence↗
Gene expression: chemical synthesis and molecular cloning of a bacteriophage T5 (T5P25) early promoter.
A sixty base pair DNA duplex containing the nucleotide sequence of the bacteriophage T5 early (T5P25) promoter has been constructed using a combination of chemical synthesis and enzymatic methods. Subsequent to cloning into pBR322, the promoter has been demonstrated to be biologically active being capable of directing the efficient expression of genes under its control. This serves as a prototype for an approach to the study of the in vivo structure-function relationships and efficiency of promoters.
Base Composition↗
Gene expression: chemical synthesis of E. coli ribosome binding sites and their use in directing the expression of mammalian proteins in bacteria.
Mammalian genes, when inserted into bacterial plasmid or phage DNAs, will not be expressed into the corresponding specific proteins in E. coli unless proper initiations signals required for recognition by E. coli ribosomes are provided. We have studied these signals and chemically synthesized two DNA duplexes each containing different initiation signals. These have been inserted in front of the Simian virus 40 (SV40) small tumor antigen gene (SV40 t gene) at varying distances from the ATG initiation codon prior to its cloning into pBR322 plasmid DNA. Plasmid containing clones carrying either of these two synthetic ribosome binding sites (RBS) at varying distances from the SV40 t gene all produced a 17K protein identical to authentic t antigen by immunologic, electrophoretic and proteolytic digestion analyses. This provides a novel method to ensure the specific expression of any contiguous mammalian gene to be cloned to bacteria, and also a unique in vivo method for studying the structure-function (efficiency) relationship of RBS with specific base changes.
Animals↗
Giant colonic diverticulum: presentation of one case and review of the literature.
Since the first case described in 1946, fewer than 100 cases of giant colonic diverticula have been reported in the literature. We discuss the clinical, radiologic, and pathologic findings in a case of giant colonic diverticulum and review the etiology and differential diagnosis.
Barium Sulfate↗
[Radiological puzzle. Intracerebral cavernous hemangioma].
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Brain Neoplasms↗
A YAC contig spanning the blepharophimosis-ptosis-epicanthus inversus syndrome and propionic acidemia loci.
Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant condition consisting of congenital dysplasia of the eyelids with a reduced horizontal diameter of the palpebral fissures, droopy eyelids and epicanthus inversus. Two clinical entities have been described: type I and type II. The former is distinguished by female infertility, whereas the latter presents without other symptoms. Both type I and type II were recently mapped on the long arm of chromosome 3 (3q22-q23), suggesting a common gene may be affected. The centromeric and the telomeric limits of this region are well defined between loci D3S1316 and D3S1615, which reside approximately 5 cM apart. Here, we present the construction of a YAC contig spanning the entire BPES locus using 17 polymorphic markers, 2 STS and 28 ESTs. This region of approximately 5 Mb was covered by 31 YACs, and was supported by detailed FISH analysis. In addition, we have precisely mapped the propionyl-CoA carboxylase beta polypeptide (PCCB), the gene mutated in propionic acidemia, within this contig. Apart from providing a framework for the identification of the BPES gene, this contig will also be useful for the future identification of defects and genes mapped to this region, and for developing template resources for genomic sequencing.
Amino Acid Metabolism, Inborn Errors↗