Decrease in GTP cyclohydrolase I gene expression caused by inactivation of one allele in hereditary progressive dystonia with marked diurnal fluctuation.

Hereditary progressive dystonia with marked diurnal fluctuation (HPD; dopa-responsive dystonia, DRD) have been recently found to be caused by a genetic defect in the GTP cyclohydrolase I (GCH1) gene. In this study, we quantified the mRNA level of GCH1 in phytohemagglutinin (PHA)-stimulated mononuclear blood cells from one Japanese family that do not have a mutation in the coding region or splice junctions of the gene. The results showed that the amounts of the GCH1 mRNA were decreased to about 40% of the normal level in both patients and carriers. In addition, we found that the GCH1 mRNA was transcribed from only one allele, indicating that the other allele was in an inactive state. These results suggest that some novel mutations should exist on one of the alleles in some unknown region of the GCH1 gene, and may decrease the GCH1 mRNA causing the HPD/DRD symptoms.     

Identification of a splice-site mutation in the aldolase B gene from an individual with hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is a potentially fatal autosomal recessive disease of carbohydrate metabolism. HFI patients exhibit a deficiency of fructose 1-phosphate aldolase (aldolase B), the isozyme expressed in tissues that metabolize fructose. The eight protein-coding exons, including splicing signals, of the aldolase B gene from one HFI patient were amplified by PCR. Dot-blot hybridization of the amplified DNA with allele-specific oligonucleotide (ASO) probes revealed a previously described A149P mutation in one allele from the proband. The mutation in the other allele was identified by direct sequencing of the double-stranded PCR-amplified material from the proband. The nucleotide sequence of exon 9 revealed a 7-base deletion/1-base insertion (delta 7 + 1) at the 3' splice site of intron 8 in one allele. This mutation was confirmed by cloning PCR-amplified exon 9 of the proband and determining the sequence of each allele separately. ASO analysis of 18 family members confirmed the Mendelian inheritance of both mutant alleles. The implications of this unique splice-site mutation in HFI are discussed.     

ApoB gene nonsense and splicing mutations in a compound heterozygote for familial hypobetalipoproteinemia.

Two novel apoB gene mutations were identified in a patient (CM) with phenotypic homozygous hypobetalipoproteinemia. Haplotype analysis of the apoB alleles from this patient and his family members revealed him to be a genetic compound for the disease. In contrast to previous studies of other hypobetalipoproteinemic patients, no clues existed as to where in the apoB gene the molecular defects resided. Therefore, it was necessary to characterize the apoB genes of the patient by sequence analysis. The apoB gene contains 29 exons and is 43 kb in length. The gene encodes a 14.1 kb mRNA and a 4563 amino acid protein. Both apoB alleles from the patient were cloned via 26 sets of polymerase chain reactions (PCR). These clones contained a total of approximately 24 kb of apoB gene sequence, including regions 5' and 3' to the coding region, 29 exons, and the intron/exon junctions. Complete DNA sequence analysis of these clones showed that each apoB allele had a mutation. In the paternal apoB allele, there was a splicing mutation. The first base of the dinucleotide consensus sequence (GT) in the 5' splice donor site in intron 5 was replaced by a T. It is likely that this base substitution interferes with proper splicing and results in the observed absence of plasma apoB. In the maternal apoB allele, there was a nonsense mutation. The first base of the Arg codon (CGA) at residue 412 in exon 10 was replaced by a T, resulting in a termination codon (TGA). The nonsense mutation is likely to terminate translation after residue 411 resulting in a severely truncated protein only 9% of the length of B-100.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential allelic expression of the type II collagen gene (COL2A1) in osteoarthritic cartilage.

Osteoarthritis (OA) is a common debilitating disease resulting from the degeneration of articular cartilage. The major protein of cartilage is type II collagen, which is encoded by the COL2A1 gene. Mutations at this locus have been discovered in several individuals with inherited disorders of cartilage. We have identified 27 primary OA patients who are heterozygous for sequence dimorphisms located in the coding region of COL2A1. These dimorphisms were used to distinguish the mRNA output from each of the two COL2A1 alleles in articular cartilage obtained from each patient. Three patients demonstrated differential allelic expression and produced < 12% of the normal level of mRNA from one of their COL2A1 alleles. The same allele shows reduced expression in all three patients, and this allele is more frequent in a well-defined OA population than in a control group, suggesting the possible existence of a rare COL2A1 allele that predisposes to OA.     

SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.     

Polarity of meiotic gene conversion is 5′ to 3′ within the niaD gene of Aspergillus nidulans

We have examined polarity of meiotic gene conversion in the niiA-niaD gene cluster of Aspergillus nidulans in two-point crosses. The type and position of the mutations represented by the niaD alleles and the correlation between the relative frequency of gene conversion and the physical position of these mutations were determined. We show that polarity of meiotic gene conversion is 5′ to 3′ (transcribed strand) within the niaD gene. Additional crosses involving a niiA allele and a niaD allele show little polarity of gene conversion, which suggests that the recombination events leading to restoration of the niaD gene are initiated upstream of the coding region of the niaD gene but within the niiA-niaD gene cluster, possibly within the intergenic promoter region.     

Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.

Fragile X syndrome results from mutations in a (CGG)n repeat found in the coding sequence of the FMR-1 gene. Analysis of length variation in this region in normal individuals shows a range of allele sizes varying from a low of 6 to a high of 54 repeats. Premutations showing no phenotypic effect in fragile X families range in size from 52 to over 200 repeats. All alleles with greater than 52 repeats, including those identified in a normal family, are meiotically unstable with a mutation frequency of one, while 75 meioses of alleles of 46 repeats and below have shown no mutation. Premutation alleles are also mitotically unstable as mosaicism is observed. The risk of expansion during oogenesis to the full mutation associated with mental retardation increases with the number of repeats, and this variation in risk accounts for the Sherman paradox.     

Heterozygosity, Heteromorphy, and Phylogenetic Trees in Asexual Eukaryotes

Little attention has been paid to the consequences of long-term asexual reproduction for sequence evolution in diploid or polyploid eukaryotic organisms. Some elementary theory shows that the amount of neutral sequence divergence between two alleles of a protein-coding gene in an asexual individual will be greater than that in a sexual species by a factor of 2tu, where t is the number of generations since sexual reproduction was lost and u is the mutation rate per generation in the asexual lineage. Phylogenetic trees based on only one allele from each of two or more species will show incorrect divergence times and, more often than not, incorrect topologies. This allele sequence divergence can be stopped temporarily by mitotic gene conversion, mitotic crossing-over, or ploidy reduction. If these convergence events are rare, ancient asexual lineages can be recognized by their high allele sequence divergence. At intermediate frequencies of convergence events, it will be impossible to reconstruct the correct phylogeny of an asexual clade from the sequences of protein coding genes. Convergence may be limited by allele sequence divergence and heterozygous chromosomal rearrangements which reduce the homology needed for recombination and result in aneuploidy after crossing-over or ploidy cycles.     

Discrimination among the transcripts of the allelic human beta-globin genes beta A, beta S and beta C using oligodeoxynucleotide hybridization probes

Three nonadecadeoxynucleotides complementary to the sense strand of the normal human beta-globin gene, beta A, and to the two allelic genes beta S and beta C were synthesized. The beta S and beta C globin genes both differ from the beta A gene by a single nucleotide substitution in the sequence coding for codon 6. The oligodeoxynucleotides are complementary to the genes in the region of the mutations and are therefore allele-specific. When radiolabeled and used as hybridization probes, the oligodeoxynucleotides are found to hybridize specifically to the mRNA transcribed from each allele.     

Reciprocal and nonreciprocal recombination at the glucocerebrosidase gene region: implications for complexity in Gaucher disease

Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocerebrosidase. The glucocerebrosidase gene is located in a gene-rich region of 1q21 that contains six genes and two pseudogenes within 75 kb. The presence of contiguous, highly homologous pseudogenes for both glucocerebrosidase and metaxin at the locus increases the likelihood of DNA rearrangements in this region. These recombinations can complicate genotyping in patients with Gaucher disease and contribute to the difficulty in interpreting genotype-phenotype correlations in this disorder. In the present study, DNA samples from 240 patients with Gaucher disease were examined using several complementary approaches to identify and characterize recombinant alleles, including direct sequencing, long-template polymerase chain reaction, polymorphic microsatellite repeats, and Southern blots. Among the 480 alleles studied, 59 recombinant alleles were identified, including 34 gene conversions, 18 fusions, and 7 downstream duplications. Twenty-two percent of the patients evaluated had at least one recombinant allele. Twenty-six recombinant alleles were found among 310 alleles from patients with type 1 disease, 18 among 74 alleles from patients with type 2 disease, and 15 among 96 alleles from patients with type 3 disease. Several patients carried two recombinations or mutations on the same allele. Generally, alleles resulting from nonreciprocal recombination (gene conversion) could be distinguished from those arising by reciprocal recombination (crossover and exchange), and the length of the converted sequence was determined. Homozygosity for a recombinant allele was associated with early lethality. Ten different sites of crossover and a shared pentamer motif sequence (CACCA) that could be a hotspot for recombination were identified. These findings contribute to a better understanding of genotype-phenotype relationships in Gaucher disease and may provide insights into the mechanisms of DNA rearrangement in other disorders.     

Differences in the nuclease sensitivity between the two alleles of the immunoglobulin kappa light chain genes in mouse liver and myeloma nuclei.

In mouse myeloma T the productive kappa light chain gene differs from its aberrantly rearranged allele in the patterns of DNAase I hypersensitive sites. In the region of the alleles where they are identical in sequence they have one site in common which lies 0.8 kb downstream of the coding region; but two sites upstream of and within the C gene segment (2) are found only on the non-productive allele. Within the region of different sequences both alleles have analogously located DNAase I hypersensitive sites; they lie 0.15 kb upstream of the respective leader segments and cover putative promoter sequences. Only one of the six DNAase I hypersensitive sites is also very sensitive towards micrococcal nuclease due to its particular DNA sequence. The non-rearranged gene studied in liver nuclei has no DNAase I hypersensitive sites but is preferentially cleaved in A/T rich regions.     

The Saccharomyces cerevisiae ADE1 gene: structure, overexpression and possible regulation by general amino acid control.

The ADE1 gene of the yeast Saccharomyces cerevisiae has been cloned by complementation of the ade1 mutation. The nucleotide sequence has been determined for the 918-bp coding region, 240-bp 5'-noncoding region and 292-bp 3'-noncoding region. The sequenced region includes a single large open reading frame coding for a protein of 306 amino acid (aa) residues. The promoter of the ADE1 gene contains a copy of the 5'-TGACTC hexanucleotide, a feature characteristic of promoters under general aa control. Subsequent search of other published purine biosynthesis gene sequences revealed that all of them also contain general aa control signals in their promoter regions. An expression plasmid containing the ADE1 coding region under control of the PHO5 promoter produced N-succinyl-5-aminoimidazole-4-carboxamide ribotide (SAICAR) synthetase in yeast cells at a level of 40% of total cellular protein. One-step purification resulted in an almost homogeneous preparation of SAICAR synthetase.