Ralph Berger

Analysis of aldehyde oxidase and xanthine dehydrogenase/oxidase as possible candidate genes for autosomal recessive familial amyotrophic lateral sclerosis

Recently, point mutations in superoxide dismutase 1 (SOD1) have been shown to lead to a subset of autosomal dominantly inherited familial amyotrophic lateral sclerosis (ALS). These findings have led to the hypothesis that defects in oxygen radical metabolism may be involved in the pathogenesis of ALS. Therefore, we decided to analyze other enzymes involved in oxygen radical metabolism for possible involvement in other forms of ALS. We report here analysis of two genes encoding the molybdenum hydroxylases aldehyde oxidase (AO) and xanthine dehydrogenase/ oxidase (XDH) for involvement in ALS. Of particular interest, one gene identified as encoding aldehyde oxidase is shown to map to 2q33, a region recently shown to contain a gene responsible for a familial form of ALS with autosomal recessive inheritance (FALS-AR). The AO gene appears to be located within 280,000 bp of simple sequence repeat marker D2S116, which shows no recombination with the FALS-AR locus. The AO gene is highly expressed in glial cells of human spinal cord. In addition, we mapped a gene for XDH to 2p22, a region previously shown to contain a highly homologous but different form of XDH. Neither of these XDH genes appears to be highly expressed in human spinal cord. This evidence suggests that AO may be a candidate gene for FALS-AR.

Refined mapping and characterization of the recessive familial amyotrophic lateral sclerosis locus (ALS2) on chromosome 2q33

ABSTRACT Amyotrophic lateral sclerosis (ALS) is a progressive degenerative neuromuscular disease that shows familial, autosomal dominant inheritance in 10%–15% of cases. Previous genetic analysis of one large family linked a recessive form of familial ALS (FALS-AR type 3) to the chromosome 2q33–35 region. Using additional polymorphic markers, we have narrowed the size of the linked region to approximately 1.7 cM by linkage and haplotype analysis. We have also established a yeast artificial chromosome contig across the locus that covers an approximate physical distance of 3 million bases. Based on this contig, genes and expressed sequences that map near the 2q33 region have been examined to determine whether they are located within this ALS2 candidate locus. Five identified genes and 34 expressed sequence tags map within the region defined by crossover analysis and merit further consideration as candidate genes for this disease.

The human type II collagen gene (COL2A1) assigned to 12q14.3

A cosmid clone containing the entire human type II α1 collagen gene (COL2A1) was used as probe in the Southern analysis of DNA from a panel of human/hamster somatic cell hybrids containing different portions of human chromosome 12. Two of the hybrids exhibited a similar terminal deletion q14.3→qter, but one was positive for the gene while the other was negative. Therefore, the gene must reside in the region q14.3.

The gene for the α2 chain of the human fibrillar collagen type XI (COL11A2) assigned to the short arm of chromosome 6

A cosmid clone (CosHcol.11) containing the α2(XI) collagen gene (COL11A2) has been isolated. The gene contains conserved DNA and amino‐acid sequences characteristic of fibril forming collagen, which is in accordance with the classification of type XI collagen as a fibrillar collagen. The genomic clone containing the α2(XI) gene has been used as probe in the Southern blot analysis of DNA from a panel of human/hamster somatic cell hybrids containing different numbers and combinations of human chromosomes. Synteny analysis revealed that only chromosome 6 showed complete concordant segregation with C0L11A2. Furthermore, the gene was regionally mapped to the short arm of chromosome 6 by using a hybrid which contained only the long arm of the chromosome.

A Single Base Change at a Splice Acceptor Site Leads to a Truncated CAD Protein in Urd-A Mutant Chinese Hamster Ovary Cells

We have previously reported the isolation and characterization of mutant Chinese hamster ovary (CHO-K1) cells of the Urd−A complementation group, which require uridine for growth, are deficient in the activities of the first three enzymes of de novo UMP biosynthesis, and produce markedly reduced amounts of a truncated form of the multifunctional protein CAD, which contains these three enzyme activities. We report here that a single base change of G to A at a highly conserved RNA splice acceptor site is responsible for the phenotype of this mutant. In addition to a small amount of apparently normal CAD mRNA, this mutation causes production of two alternative forms of CAD mRNA in the mutant, one that includes the intron just prior to the mutation and one that excludes the exon just after the mutation. The affected splice site is located at the intron-exon boundary just preceding the exon that encodes the beginning of the aspartate transcarbamylase (ATCase) domain of the CAD protein. Both intron inclusion and exon exclusion during RNA processing introduce a translation stop codon upstream of the region encoding this domain, resulting in the production of the truncated CAD protein seen in the Urd−A mutant. This mutation also results in markedly decreased levels of CAD mRNA and protein in the mutant.

Molecular analysis of human repetitive sequence family and its use as genetic marker

It has been known for over 15 years that the human genome consists of roughly 65% single or low-copy-number DNA sequences and 35% repeated sequences (1). Since then, the discovery of restriction endonucleases and the development of molecular cloning and DNA sequencing techniques have made it possible to study these sequences at the molecular level. Most gene sequences are represented by single-copy DNA, and the cloning and nucleotide sequencing of these genes have proven to be very rewarding and highly informative. Similar studies involving repetitive sequences have also been carried out in many laboratories; however, much of the information gathered to date has been structural, leaving the functions of repetitive sequences a matter of speculation. Repetitive sequences are usually categorized into two major classes, the highly repetitive sequences and the moderately repetitive sequences. The highly repetitive sequences have over 106 copies per haploid genome, some of which are dispersed while most are clustered at centromeres and telomeres. This group of clustered sequences is often known as satellite DNA, and its function remains unknown (2). For the most part, satellite DNA is not transcribed into RNA, and in those rare instances where it is, it might be the result of readthrough from the transcription of legitimate genes, such as histones, which are embedded in repeated sequences (3, 4). The second class, the middle or moderately repetitive sequences, is much more complex than the first class. It ranges from a few hundred copies to hundreds of thousands of copies in the genome. These sequences are usually scattered throughout the genome next to unique or other repetitive sequences (5) and sometimes even interrupting satellite DNA sequences (6). Most of the progress made in the study of this complex class of DNA has resulted from the application of modern techniques in molecular genetics. One distinction that can be made when looking at these dispersed repetitive sequences as a whole is the length of the repeat unit. Singer (7) suggested the notation SINES for short interspersed repeated segments, usually 500 bp or less in length and present in as many as hundreds of thousands of copies in the mammalian genome, and the notat ion L I N E S for long interspersed repeated segments, usually several kilobases (kb) in length and repeated perhaps 104 times or less in the genome. The best character ized mammalian SINE family is the human Alu family (8, 9) which has 3 • 105 copies in the haploid genome. Although the structure of this family has been well characterized, its function remains elusive. The finding that the Alu sequences share homology with a low-molecular-weight RNA species, 7S RNA, suggests that this repeat family may play a role in protein secretion, because 7S RNA is a component of the 11S signal recognition particle