Normal and expanded Huntington's disease gene alleles produce distinguishable proteins due to translation across the CAG repeat.

BACKGROUND: An expanded CAG trinucleotide repeat is the genetic trigger of neuronal degeneration in Huntington's disease (HD), but its mode of action has yet to be discovered. The sequence of the HD gene places the CAG repeat near the 5' end in a region where it may be translated as a variable polyglutamine segment in the protein product, huntingtin. MATERIALS AND METHODS: Antisera directed at amino acid stretches predicted by the DNA sequence upstream and downstream of the CAG repeat were used in Western blot and immunohistochemical analyses to examine huntingtin expression from the normal and the HD allele in lymphoblastoid cells and postmortem brain tissue. RESULTS: CAG repeat segments of both normal and expanded HD alleles are indeed translated, as part of a discrete approximately 350-kD protein that is found primarily in the cytosol. The difference in the length of the N-terminal polyglutamine segment is sufficient to distinguish normal and HD huntingtin in a Western blot assay. CONCLUSIONS: The HD mutation does not eliminate expression of the HD gene but instead produces an altered protein with an expanded polyglutamine stretch near the N terminus. Thus, HD pathogenesis is probably triggered by an effect at the level of huntingtin protein.     

A gene encoding a fibroblast growth factor receptor isolated from the Huntington disease gene region of human chromosome 4

The gene responsible for Huntington disease (HD), an autosomal dominant neurodegenerative disorder, is located near the terminus of the short arm of chromosome 4. Detailed genetic linkage and physical mapping studies have defined a region of approximately 2.5 million basepairs where the disease gene is likely to be located. Efforts to identify the disease gene are now focused on the identification and characterization of expressed genes in this region. Nucleotide sequence analysis of a cDNA clone derived from the HD gene region has revealed that it encodes a member of the fibroblast growth factor subfamily of tyrosine kinase receptors, some members of which are known to be involved in the differentiation and survival of certain cell types within the central nervous system. Histochemical analysis using in situ hybridization revealed its expression in many areas of the brain, among them being the caudate and putamen. The nature of this gene, FGFR3, and its map location make it a possible candidate for the HD gene.     

A new DNA marker (D4S90) is located terminally on the short arm of chromosome 4, close to the Huntington disease gene

Genetic linkage studies have mapped Huntington's disease (HD) to the distal portion of the short arm of chromosome 4 (4p16.3), 4 cM distal to D4S10 (G8). To date, no definite flanking marker has been identified. A new DNA marker, D4S90 (D5); which maps to the distal region of 4p16.3, is described. The marker was used in a genetic linkage study in the CEPH reference families with seven other markers at 4p16. The study, together with knowledge of the physical map of the region, places D4S90 as the most distal marker, 6 cM from D4S10. A provisional linkage study with HD gave a maximum lod score of 2.14 at a theta of 0.00 and no evidence of linkage disequilibrium. As D4S90 appears to be located terminally, it should play an important role in the accurate mapping and cloning of the HD gene.     

A yeast artificial chromosome telomere clone spanning a possible location of the Huntington disease gene

The Huntington disease (HD) gene has been mapped to the most distal subband of chromosome 4p. Analysis of recombination events has not provided an unequivocal location of the HD gene, but it indicates a position very close to the telomere as one possibility. We have constructed a yeast artificial chromosome (YAC) vector (containing a rare-cutter polylinker) for the cloning of mammalian telomeres, used it to prepare a BssHII-telomere library with DNA from an individual homozygous for HD, and have identified a 115-kb clone containing the telomere of 4p. One probable recombinant would confine the telomeric candidate location for the gene to the region covered by the YAC, which makes it possible that the clone described here contains the HD locus in its mutant form.     

Construction of a NotI linking library and isolation of new markers close to the Huntington''s disease gene.

Linking clones contain sequences flanking recognition sites for enzymes cutting rarely in mammalian DNA. They can be used to obtain and correlate both physical and genetic mapping information over subregions of mammalian chromosomes. We have constructed and used a NotI linking clone library representing unmethylated NotI sites from HHW693 DNA, a hamster hybrid cell line containing 4p15-4pter and a fragment of 5p as its only human chromosome contribution. Human clones were identified by hybridisation with a cloned human repeat sequence, and localised further to subregions of human chromosome 4p15-4pter using a panel of additional hybrids. Clones from the region distal to the DNA probes (D4S10, D4S43, D4S95) linked to the Huntington's disease mutation, were further analysed. Four markers close to the HD gene: D4S111, D4S113, D4S114 and clone 417 are described here. In addition to serving as markers in physical and genetic mapping experiments, these linking clones provide probes next to cleavable NotI sites, and can therefore be used to screen NotI based chromosome jumping libraries. They also provide indications for potential gene sequences, identifiable as evolutionarily conserved sequences.     

Evidence from family studies that the gene causing Huntington disease is telomeric to D4S95 and D4S90.

A DNA probe (D4S95) that detects a variable number of tandem repeats and a single-site-variation polymorphism after digestion with a single restriction enzyme, AccI, has previously been described. The order of this probe relative to the gene for Huntington disease (HD) and other previously described markers has not been established. Analysis of 24 affected families with HD has shown that D4S95 is in tight linkage with the gene causing HD, with a maximal Lod score of 12.489 at a theta of .03. D4S90 is a probe which maps to 4p16.3, telomeric to D4S95, and detects polymorphisms with HincII and other enzymes. In one affected person, recombination has occurred between D4S10 and HD, between D4S95 and HD, and in all likelihood also between D4S90 and HD, which strongly suggests that the gene for HD is telomeric to all these DNA probes. This suggests that the gene causing HD is located in the most distal region of the short arm of chromosome 4, flanked by D4S90 and the telomere, and supports the locus order D4S10-D4S95-D4S90-HD-telomere. D4S95 is a most useful DNA marker for predictive testing programs, while D4S90 will serve as a useful starting point for identifying DNA fragments closer to the gene for HD.     

Increased recombination adjacent to the Huntington disease-linked D4S10 marker.

Huntington disease (HD) is caused by a genetic defect distal to the anonymous DNA marker D4S10 in the terminal cytogenetic subband of the short arm of chromosome 4 (4p16.3). The effort to identify new markers linked to HD has concentrated on the use of somatic cell hybrid panels that split 4p16.3 into proximal and distal portions. Here we report two new polymorphic markers in the proximal portion of 4p16.3, distal to D4S10. Both loci, D4S126 and D4S127, are defined by cosmids isolated from a library enriched for sequences in the 4pter-4p15.1 region. Physical mapping by pulsed-field gel electrophoresis places D4S126 200 kb telomeric to D4S10, while D4S127 is located near the more distal marker D4S95. Typing of a reference pedigree for D4S126 and D4S127 and for the recently described VNTR marker D4S125 has firmly placed these loci on the existing linkage map of 4p16.3. This genetic analysis has revealed that the region immediately distal to D4S10 shows a dramatically higher rate of recombination than would be expected based on its physical size. D4S10-D4S126-D4S125 span 3.5 cM, but only 300-400 kb of DNA. Consequently, this small region accounts for most of the reported genetic distance between D4S10 and HD. By contrast, it was not possible to connect D4S127 to D4S125 by physical mapping, although they are only 0.3 cM apart. A more detailed analysis of recombination sites within the immediate vicinity of D4S10 could potentially reveal the molecular basis for this phenomenon; however, it is clear that the rate of recombination is not continuously increased with progress toward the telomere of 4p.     

Isolation of DNA markers in the direction of the Huntington disease gene from the G8 locus.

To facilitate identification of additional DNA markers near and on opposite sides of the Huntington disease (HD) gene, we developed a panel of somatic-cell hybrids that allows accurate subregional mapping of DNA fragments in the distal portion of 4p. By means of the hybrid-cell mapping panel and a library of DNA fragments enriched for sequences from the terminal one-third of the short arm of chromosome 4, 105 DNA fragments were mapped to six different physical regions within 4p15-4pter. Four polymorphic DNA fragments of particular interest were identified, at least three of which are distal to the HD-linked D4S10 (G8) locus, a region of 4p previously devoid of DNA markers. Since the HD gene has also recently been shown to be distal to G8, these newly identified DNA markers are in the direction of the HD gene from G8, and one or more of them may be on the opposite side of HD from G8.     

Chromosome mapping of the rod photoreceptor cGMP phosphodiesterase beta-subunit gene in mouse and human: tight linkage to the Huntington disease region (4p16.3).

The retinal degeneration mouse (gene symbol, rd) is an animal model for certain forms of human hereditary retinopathies. Recent findings of a nonsense mutation in the rd mouse PDE beta-subunit gene (Pdeb) prompted us to investigate the chromosome locations of the mouse and human genes. We have utilized backcross analysis in mice to verify and define more precisely the location of the Pdeb locus 6.1 +/- 2.3 cM distal of Mgsa on mouse chromosome 5. We have determined that the human gene (PDEB) maps to 4p16.3, very close to the Huntington disease (HD) region. Analysis of the comparative map for mice and humans shows that the mouse homologue of the HD gene will reside on chromosome 5. Linkage of the mouse Pdeb locus with other homologues in the human 4p16.3 region is maintained but gene order is not, suggesting at least three possible sites for the corresponding mouse HD gene.     

Nucleotide Sequence of the Akv env Gene

The sequence of 2,191 nucleotides encoding the env gene of murine retrovirus Akv was determined by using a molecular clone of the Akv provirus. Deduction of the encoded amino acid sequence showed that a single open reading frame encodes a 638-amino acid precursor to gp70 and p15E. In addition, there is a typical leader sequence preceding the amino terminus of gp70. The locations of potential glycosylation sites and other structural features indicate that the entire gp70 molecule and most of p15E are located on the outer side of the membrane. Internal cleavage of the env precursor to generate gp70 and p15E occurs immediately adjacent to several basic amino acids at the carboxyl terminus of gp70. This cleavage generates a region of 42 uncharged, relatively hydrophobic amino acids at the amino terminus of p15E, which is located in a position analogous to the hydrophobic membrane fusion sequence of influenza virus hemagglutinin. The mature polypeptides are predicted to associate with the membrane via a region of 30 uncharged, mostly hydrophobic amino acids located near the carboxyl terminus of p15E. Distal to this membrane association region is a sequence of 35 amino acids at the carboxyl terminus of the env precursor, which is predicted to be located on the inner side of the membrane. By analogy to Moloney murine leukemia virus, a proteolytic cleavage in this region removes the terminal 19 amino acids, thus generating the carboxyl terminus of p15E. This leaves 15 amino acids at the carboxyl terminus of p15E on the inner side of the membrane in a position to interact with virion cores during budding. The precise location and order of the large RNase T(1)-resistant oligonucleotides in the env region were determined and compared with those from several leukemogenic viruses of AKR origin. This permitted a determination of how the differences in the leukemogenic viruses affect the primary structure of the env gene products.     

Recombination events suggest potential sites for the Huntington's disease gene

The Huntington's disease gene (HD) maps distal to the D4S10 marker in the terminal 4p16.3 subband of chromosome 4. Directed cloning has provided several DNA segments that have been grouped into three clusters on a physical map of approximately 5 X 10(6) bp in 4p16.3. We have typed RFLPs in both reference and HD pedigrees to produce a fine-structure genetic map that establishes the relative order of the clusters and further narrows the target area containing the HD gene. Despite the large number of meiotic events examined, the HD gene cannot be positioned relative to the most distal cluster. One recombination event with HD suggests that the terminal-most markers flank the disease gene; two others favor a telomeric location for the defect. Efforts to isolate the HD gene must be divided between these two distinct intervals until additional genetic data resolve the apparent contradiction in localization.     

Isolation and field-inversion gel electrophoresis analysis of DNA markers located close to the Huntington disease gene

A radiation-induced hybrid cell line containing 10-20 million base pairs of DNA derived from the terminal part of human 4p16 in a background of hamster chromosomes has been used to construct a genomic library highly enriched for human sequences located close to the Huntington disease (HD) gene. Recombinant phage containing human inserts were isolated from this library and used as hybridization probes against two other radiation hybrids containing human fragments with chromosomal breaks in 4p16 and against a human-hamster somatic cell hybrid that retains only the 4p15-4pter part of chromosome 4. Of 121 human phage tested, 6 were mapped distal to the HD-linked D4S10 locus. Since the HD gene is located between D4S10 and the 4p telomere, all of these sequences are likely to be closer to HD than D4S10, and any one of them may be a distal flanking marker for the disease locus. Long-range restriction map analysis performed with a field-inversion gel system shows that the six new loci are distributed in different places within 4p16. Although it is not possible to establish an order for the six sequences with the FIGE data, the results demonstrate that the region detected by these probes must span at least 2000 kb of DNA.     

The Saccharomyces cerevisiae chromosome III left telomere has a type X, but not a type Y'', ARS region.

A yeast Saccharomyces cerevisiae telomeric region was isolated by chromosome walking from HML alpha, the most distal known gene on the chromosome III left (IIIL) end. The terminal heterodisperse 3.3-kilobase (kb) SalI fragment on chromosome IIIL, 8.6 kb distal to HML alpha, was cloned in a circular vector to generate a telomeric probe. Southern hybridization and DNA sequencing analyses indicated that 0.6 kb (+/- 200 base pairs) of 5'-C1-3A-3' simple tandem repeat sequence, adjacent to a 1.2-kb type X ARS region, constitutes the telomere on the chromosome IIIL end, and no type Y' ARS region homologies exist between HML alpha and the IIIL terminus.     

Isolation of novel herpes simplex virus type 1 derivatives with tandem duplications of DNA sequences encoding immediate-early mRNA-5 and an origin of replication.

Two naturally occurring variations of herpes simplex virus type 1 (Patton strain) with novel tandem DNA sequence duplications in the S component were isolated, and the DNA was characterized. These variants were identified among a number of plaque isolates by the appearance of new restriction enzyme fragments that hybridized with radiolabeled DNA from the BamHI Z fragment (map coordinates 0.936 to 0.949) located in the unique S region. One isolate, SP26-3, carried a 3.1-kilobase-pair duplication defined by recombination between a site in the BamHI Z fragment and a site near the origin of replication in the inverted repeat sequence of the S component carried by the EcoRI H fragment. The other isolate, SP22-4, carried a 3.5-kilobase-pair duplication defined by a recombination event between a tandem repeat array in the BamHI Z fragment and a site near the amino terminus of the Vmw175 gene in the S-region inverted repeat sequence contained in the EcoRI K fragment. Both duplicated segments contained the entire immediate early mRNA-5 coding region as well as the origin of replication located in the inverted repeat sequence of the S component. The DNA sequence of each duplication joint was determined.     

棉铃虫核多角体病毒p10基因的序列及转录分析

为了利用棉铃虫核多角体病毒（HaSNPV）p10基因的启动子构建重组病毒杀虫剂及表达系统,应用末端测序法和引物步移法测定了p10基因的序列,并应用Northern杂交和引物延伸实验对该基因进行了深入的转录特性分析,HaSNPV p10基因被定位于基因组DNA的115.4kb-115.6kb处,转录方向与多角体基因相反,在其上下游分别发现了p26和p74基因。转录分析结果确定了p10基因是个极晚期基因,其转录从杆状病毒晚期转录保守序列GTAAG的第二个A开始,转录产物大小约为430nt。HaSNPV p10基因最早可检测到的转录是在病毒感染后的20h,随后其转录产物量迅速放大,到感染后72h达到非常高的水平。围康时相分析同时还检测到一个大小为1300nt的产物,推测该产物为p26和p10通读的转录产物。对HaSNPV P10蛋白序列的分析表明,该蛋白第6－44位及第51－65位氨基酸序列处含多个典型的卷曲螺旋七聚体重复结构,两片段间相隔6个氨基酸残基,在该蛋白序列的第20－34位与第51－65位存在L－X（2）－L－X（10）－L的亮氨酸重复。Helical net分析表明,HaSNPV P10的疏水氨式酸分布为两个集簇区,两者互为180度转角。     

Chromosome mapping of the rod photoreceptor cGMP phosphodiesterase β-subunit gene in mouse and human: Tight linkage to the Huntington disease region (4p16.3)

The retinal degeneration mouse (gene symbol, rd) is an animal model for certain forms of human hereditary retinopathies. Recent findings of a nonsense mutation in the rd mouse PDE β-subunit gene (Pdeb) prompted us to investigate the chromosome locations of the mouse and human genes. We have utilized backcross analysis in mice to verify and define more precisely the location of the Pdeb locus 6.1 ± 2.3 cM distal of Mgsa on mouse chromosome 5. We have determined that the human gene (PDEB) maps to 4p16.3, very close to the Huntington disease (HD) region. Analysis of the comparative map for mice and humans shows that the mouse homologue of the HD gene will reside on chromosome 5. Linkage of the mouse Pdeb locus with other homologues in the human 4p16.3 region is maintained but gene order is not, suggesting at least three possible sites for the corresponding mouse HD gene.     

A new DNA marker (D4S90) is located terminally on the short arm of chromosome 4, close to the Huntington disease gene

Genetic linkage studies have mapped Huntington's disease (HD) to the distal portion of the short arm of chromosome 4 (4p16.3), 4 cM distal to D4S10 (G8). To date, no definite flanking marker has been identified. A new DNA marker, D4S90 (D5), which maps to the distal region of 4p16.3, is described. The marker was used in a genetic linkage study in the CEPH reference families with seven other markers at 4p16. The study, together with knowledge of the physical map of the region, places D4S90 as the most distal marker, 6 cM from D4S10. A provisional linkage study with HD gave a maximum lod score of 2.14 at a θ of 0.00 and no evidence of linkage disequilibrium. As D4S90 appears to be located terminally, it should play an important role in the accurate mapping and cloning of the HD gene.     

Provirus of M7 baboon endogenous virus: nucleotide sequence of the gag-pol region

A 3,023-base nucleotide sequence of the M7 baboon endogenous virus genome, spanning the 5' noncoding region as well as the entire gag gene and part of the pol gene, is reported. Within the 562-base 5' noncoding region, a 21-base sequence complementary to the OH terminus of tRNApro is located immediately downstream from the long terminal repeat. Amino acid sequences were deduced from the 1,596 nucleotides comprising the gag gene, and the four structural gag polypeptides, p12, p15, p30, and p10, appeared to be coded contiguously. Only one termination codon interrupted the M7 gag and pol genes. The data suggest that 55 additional amino acids may be attached to the NH2 terminus of the gag precursor protein. However, such a sequence was not detected in virions or in virus-infected cells. With the exception of the p15 region, nucleotide and amino acid sequences of the gag and pol regions of M7 virus exhibited strong homologies to those of Moloney leukemia virus.