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R L Nussbaum

Publications and source records attributed to R L Nussbaum.

At least 55 records · Page 3Linked to original sources
PubMed1995

FXR1, an autosomal homolog of the fragile X mental retardation gene.

Fragile X mental retardation syndrome, the most common cause of hereditary mental retardation, is directly associated with the FMR1 gene at Xq27.3. FMR1 encodes an RNA binding protein and the syndrome results from lack of expression of FMR1 or expression of a mutant protein that is impaired in RNA binding. We found a novel gene, FXR1, that is highly homologous to FMR1 and located on chromosome 12 at 12q13. FXR1 encodes a protein which, like FMR1, contains two KH domains and is highly conserved in vertebrates. The 3' untranslated regions (3'UTRs) of the human and Xenopus laevis FXR1 mRNAs are strikingly conserved (approximately 90% identity), suggesting conservation of an important function. The KH domains of FXR1 and FMR1 are almost identical, and the two proteins have similar RNA binding properties in vitro. However, FXR1 and FMR1 have very different carboxy-termini. FXR1 and FMR1 are expressed in many tissues, and both proteins, which are cytoplasmic, can be expressed in the same cells. Interestingly, cells from a fragile X patient that do not have any detectable FMR1 express normal levels of FXR1. These findings demonstrate that FMR1 and FXR1 are members of a gene family and suggest a biological role for FXR1 that is related to that of FMR1.

Amino Acid Sequence↗
PubMed1995

Nondisjunction rates and abnormal embryonic development in a mouse cross between heterozygotes carrying a (7, 18) robertsonian translocation chromosome.

Mice bearing Robertsonian translocation chromosomes frequently produce aneuploid gametes. They are therefore excellent tools for studying nondisjunction in mammals. Genotypic analysis of embryos from a mouse cross between two different strains of mice carrying a (7,18) Robertsonian chromosome enabled us to measure the rate of nondisjunction for chromosomes 7 and 18. Embryos (429) were harvested from 76 litters of mice and the parental origin of each chromosome 7 and 18 determined. Genotyping these embryos has allowed us to conclude the following: (1) there were 96 embryos in which at least one nondisjunction event had taken place; (2) the rate of maternal nondisjunction was greater than paternal nondisjunction for teh chromosomes sampled in these mice; (3) a bias against chromosome 7 and 18 nullisomic gametes was observed, reflected in a smaller than expected number of uniparental disomic embryos; (4) nondisjunction events did not seem to occur at random throughout the 76 mouse litters, but were clustered into fewer than would be expected cy chance; and (5) a deficiency of paternal chromosome 18 uniparental disomic embryos was observed along with a higher than normal rate of developmental retardation at 8.5 days post coitum, raising the possibility that this chromosome has at least one imprinted gene.

Aneuploidy↗
PubMed1995

The oculocerebrorenal syndrome gene product is a 105-kD protein localized to the Golgi complex.

The oculocerebrorenal syndrome of Lowe (OCRL) is a multisystem disorder affecting the lens, kidney, and CNS. The predicted amino acid sequence of the OCRL gene, OCRL-1, was used to develop antibodies against the OCRL-1 protein. Western blot analysis using affinity-purified serum against the amino terminus of the OCRL-1 gene product (ocrl-1) demonstrates a single protein of 105 kD in fibroblasts of a normal individual that is absent in fibroblasts of an OCRL patient who lacks OCRL-1 transcript. A single protein with the same electrophoretic mobility is found by western analysis in various human cultured cell lines, and approximately the same size protein is also found in all mouse tissues tested. Northern analysis of various human and mouse tissues demonstrate that OCRL-1 transcript is expressed in nearly all tissues examined. By immunofluorescence, the ocrl-1 antibody stains a juxtanuclear region in normal fibroblast cells, while no specific staining is evident in the OCRL patient who produces no transcript. Colocalization of the ocrl-1 protein to the Golgi complex was demonstrated using a known monoclonal antibody against a Golgi-specific coat protein, beta-COP (beta coatomer protein).

Adult↗
PubMed1994

Essential role for KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome.

The KH domain is an evolutionarily conserved sequence motif present in many RNA-binding proteins, including the pre-mRNA-binding (hnRNP) K protein and the fragile X mental retardation gene product (FMR1). We assessed the role of KH domains in RNA binding by mutagenesis of KH domains in hnRNP K and FMR1. Conserved residues of all three hnRNP K KH domains are required for its wild-type RNA binding. Interestingly, while fragile X syndrome is usually caused by lack of FMR1 expression, a previously reported mutation in a highly conserved residue of one of its two KH domains (Ile-304-->Asn) also results in mental retardation. We found that the binding of this mutant protein to RNA is severely impaired. These results demonstrate an essential role for KH domains in RNA binding. Furthermore, they strengthen the connection between fragile X syndrome and loss of the RNA binding activity of FMR1.

Amino Acid Sequence↗
PubMed1994

Isolation and regional mapping of 110 chromosome 22 STSs.

As part of a larger effort to create a complete physical map of the human genome, we have developed 110 new STSs specific for human chromosome 22. Clones isolated and sequenced from chromosome 22-enriched libraries provided a source of primers. These STSs were localized to regions of chromosome 22 using a panel of somatic cell hybrids. In building a refined physical map of chromosome 22, this set of STSs should provide a substantial backbone.

Animals↗
PubMed1994

Localization of the 75-kDa inositol polyphosphate-5-phosphatase (INPP5B) to human chromosome band 1p34.

The 75-kDa (type II) inositol polyphosphate-5-phosphatase, originally described in platelets, is one of at least three known enzymes capable of dephosphorylating inositol-1,4,5-trisphosphate (IP3) to inositol-1,4-bisphosphate (IP2). To further characterize these enzymatic forms, we have mapped the gene (INPP5B) coding for the 75-kDa type II enzyme. Using a combination of human x rodent somatic cell hybrids and fluorescence in situ hybridization, we have determined that this gene maps to human chromosome band 1p34.

Animals↗
PubMed1993

The protein product of the fragile X gene, FMR1, has characteristics of an RNA-binding protein.

Fragile X syndrome is one of the most common human genetic diseases and the most common cause of hereditary mental retardation. The gene that causes fragile X syndrome, FMR1, was recently identified and sequenced and found to encode a putative protein of unknown function. Here we report that FMR1 contains two types of sequence motifs recently found in RNA-binding proteins: an RGG box and two heterogeneous nuclear RNP K homology domains. We also demonstrate that FMR1 binds RNA in vitro. Using antibodies to FMR1, we detect its expression in divergent organisms and in cells of unaffected humans, but fragile X-affected patients express little or no FMR1. These findings demonstrate that FMR1 expression is directly correlated with the fragile X syndrome and suggest that anti-FMR1 antibodies will be important for diagnosis of fragile X syndrome. Furthermore, the RNA binding activity of FMR1 opens the way to understanding the function of FMR1.

Amino Acid Sequence↗
PubMed1993

Deletion mapping and a highly reduced radiation hybrid in the Xp22.3-p22.2 region.

The human Xp22.3-p22.2 region contains several known disease genes as well as distinctive families of low copy repetitive sequences. In this study, we have developed new tools to more finely map this area. We have characterized a mapping panel of various cell lines and hybrids with different molecular breakpoints as defined by previously mapped reference markers from this region. The panel subdivides this area into nine distinct regions from DXS41 through the pseudoautosomal boundary. We have also identified a radiation-reduced somatic cell hybrid, Z4-7, that contains DXS31, DXS452, STS, DXS143, and DXS85, but not PABX, DXS16, or other single-copy probes from proximal Xp and Xq. A phage library was constructed from Z4-7 and over 80,000 plaques were screened with total human DNA. More than 100 positive clones were identified as potential new markers in this region. Nine of these have been mapped to the hybrid panel, and unique subclones have been isolated from three of these markers. The panel has also allowed us to map several other DNA markers, genes (AMG, OA1), and repetitive elements of the DXF22S and DXF30S sequence families relative to the various breakpoints.

Albinism, Ocular↗
PubMed1993

Nonsense mutations in the OCRL-1 gene in patients with the oculocerebrorenal syndrome of Lowe.

A candidate gene, OCRL-1, for the oculocerebrorenal syndrome of Lowe (OCRL) has been identified via positional cloning strategies. We have now developed RT-PCR techniques which allow amplification of nearly all of the open reading frame from total RNA and have used the PCR products for mutational analysis. Single strand conformational polymorphism analysis detected aberrant migration in two unrelated patients, both of whom were shown to have the same nonsense mutation at base 2746 on direct sequencing. An additional patient was found to be missing a segment from his RNA that corresponds to an entire exon. The identification of mutations in the OCRL-1 gene provides strong genetic evidence for its being the gene involved in Lowe syndrome.

Adult↗
PubMed1992

Cloning of human and bovine homologs of SNF2/SWI2: a global activator of transcription in yeast S. cerevisiae.

We performed positional cloning of genes carried on yeast artificial chromosomes that span a human translocation breakpoint associated with a human disease and isolated by chance human and bovine genes with strong homology to the S. cerevisiae genes, SNF2/SWI2 and STH1, and the D. melanogaster gene brahma. We report here sequence analysis, expression data, and functional studies for this human SNF2-like gene (hSNF2L) and its bovine homolog (bovSNF2L). Despite strong homology at the amino acid level, hSNF2L is not capable of complementing the yeast mutations snf2 or sth1 in S. cerevisiae. Furthermore, in contrast to SNF2 itself, a fusion protein consisting of the DNA binding domain of LexA and hSNF2L did not transactivate a reporter gene downstream of LexA binding sites in a yeast expression system. The strong similarity between hSNF2L and these yeast and drosophila genes suggest that the mammalian genes are part of an evolutionarily conserved family that has been implicated as global activators of transcription in yeast and fruitflies but whose function in mammals remains unknown.

Adenosine Triphosphatases↗
PubMed1992

The Lowe's oculocerebrorenal syndrome gene encodes a protein highly homologous to inositol polyphosphate-5-phosphatase.

Lowe's oculocerebrorenal syndrome (OCRL) is a human X-linked developmental disorder of unknown pathogenesis and has a pleiotropic phenotype affecting the lens, brain and kidneys. The OCRL locus has been mapped to Xq25-q26 by linkage and by finding de novo X; autosome translocations at Xq25-q26 in two unrelated females with OCRL. Here we use yeast artificial chromosomes with inserts that span the X chromosomal breakpoint from a female OCRL patient in order to isolate complementary DNAs for a gene that is interrupted by the translocation. We show that the transcript is absent in both female OCRL patients with X; autosome translocations and that it is absent or abnormally sized in 9 of 13 unrelated male OCRL patients with no detectable genomic rearrangement. The open reading frame encodes a new protein with 71% similarity to human inositol polyphosphate-5-phosphatase. Our results suggest that OCRL may be an inborn error of inositol phosphate metabolism.

Amino Acid Sequence↗
PubMed1992

Isolation of a candidate gene for choroideremia.

Choroideremia is an X chromosome-linked retinal dystrophy of unknown pathogenesis. We have isolated cDNAs from a human retinal library with a genomic probe located at the X chromosomal breakpoint in a female with choroideremia and an X;13 translocation. This cDNA spans the breakpoint in the X;13 translocation female and is deleted in males who have choroideremia as part of a complex phenotype including mental retardation and deafness. However, this cDNA detects no alterations in the DNA of 34 males with isolated choroideremia. Nonetheless, the cDNA does detect reduced or absent levels of mRNA in three-quarters of male patients with an apparently intact gene. These data support the hypothesis that this cDNA represents the gene in which mutations cause choroideremia.

Amino Acid Sequence↗
PubMed1992

Isolation of cDNA sequences around the chromosomal breakpoint in a female with Lowe syndrome by direct screening of cDNA libraries with yeast artificial chromosomes.

The Lowe oculocerebrorenal syndrome (OCRL; McKusick 309000) is an X-linked disorder characterized by congenital cataracts, muscular hypotonia, mental retardation, and Fanconi syndrome of the renal tubules. A pair of yeast artificial chromosomes (YACs) that span the Xq25-q26 translocation breakpoint in a female with OCRL were used as probes to screen cDNA libraries made from bovine lens and human kidney. The methods used to prepare the YACs as probes and to screen the libraries are presented in detail. Two different transcripts were found that map to the region around the Xq25-q26 breakpoint. These transcripts are now being studied to determine whether one or the other is a candidate gene for OCRL.

Blotting, Southern↗
PubMed1992

Construction and characterization of a yeast artificial chromosome library for Xpter-Xq27.3: a systematic determination of cocloning rate and X-chromosome representation.

We describe the construction and characterization of a human X-chromosome-specific yeast artificial chromosome (YAC) library. Starting with 60 micrograms of hybrid cell line genomic DNA, we generated over 150,000 recombinants, over 90% of which range from 150 to 500 kb. From these recombinants, 3300 human-positive YACs (representing coverage of 4.5 X chromosomes) were identified by genomic human DNA hybridization. Mapping of random clones revealed that they are derived from the X chromosome in a regionally unbiased fashion, and screening with single-copy X-chromosome probes has repeatedly produced YACs from the library. By determining the frequency of YAC clones containing both hamster and human repetitive sequences, we estimated that approximately 11% of clones contain discontiguous sequences. Taken together, the low cocloning rate, the unbiased coverage, and a consistent recovery of YACs using specific X-chromosome markers indicate that YAC technology can be used for extensive cloning and mapping purposes. Because a certain amount of genomic rearrangement is present in YAC libraries, chromosome walking must be undertaken with a degree of caution.

Animals↗
PubMed1992

Maximum-likelihood analysis of human T-cell X chromosome inactivation patterns: normal women versus carriers of X-linked severe combined immunodeficiency.

Lymphocytes of female carriers of X-linked severe combined immunodeficiency (XSCID; McKusick 300400; HGM genetic locus designation SCIDX1) exhibit nonrandom X chromosome inactivation. This phenomenon reflects a tissue-specific selective disadvantage for lymphocyte progenitors with an XSCID mutation on the active X chromosome and presumably is analogous to the process that inhibits T-cell development in affected boys with a single XSCID-bearing X chromosome. We investigated the specificity of T-cell X chromosome inactivation pattern as an indicator of immunodeficiency carrier status, as follows: X-inactivation ratios determined in a control group of noncarrier women exhibited a wide range, 20%-86% of T-cells with the paternal X active. Maximum-likelihood analysis of these data suggested that, in humans, mature T-cells are derived from a small pool of only about 10 randomly inactivated stem cells. Despite the wide variability in normal X-inactivation ratios, X inactivation in XSCID carriers appeared far more markedly skewed. Therefore a maximum-likelihood odds-ratio test was developed and proved to be successful in predicting the carrier status of women in XSCID pedigrees. This test has made it possible to identify XSCID carriers among mothers of boys with the heterogeneous syndrome of sporadic severe combined immunodeficiency.

Adolescent↗
PubMed1991

Linkage analysis in X-linked ocular albinism.

We studied the linkage of X-linked Nettleship-Falls ocular albinism (OA1) to Xp22.1-Xp22.3 RFLPs at 12 loci in five families, including one in which OA1 cosegregates with a deletion of steroid sulfatase (STS). We found evidence for tight linkage of OA1 to the Xp22.3 loci DXS143, STS, and DXS452. DXS452, a newly described polymorphism detected by the probe E25B1.8, is part of the sequence family "DXS278" (pCRI-S232), but represents a single genetic locus. Every female in this study was heterozygous for the DXS452 RFLP. Thus, this marker will be extremely useful for family studies and genetic counseling. Analysis of individual recombinations suggests that OA1 maps between DXS143 and DXS85. Multipoint linkage analysis was consistent with this localization but was not statistically significant. These data suggest that OA1 lies proximal to the deletion in a previously described family with OA1 and STS deletion, but maps within the Xp22.3-Xp22.2 region.

Albinism, Ocular↗
PubMed1991

In situ hybridization to cytogenetic bands of yeast artificial chromosomes covering 50% of human Xq24-Xq28 DNA.

From the collection described by Abidi et al., 102 yeast artificial chromosomes (YACs) with human DNA inserts more than 300 kb in length were assigned to chromosomal band positions on early metaphase chromosomes by in situ hybridization using the biotin-avidin method. All the YACs hybridized within the Xq24-Xqter region, supporting the origin of the vast majority of the YACs from single human X-chromosomal sites. With assignments precise to +/- 0.5 bands, YACs were distributed among cytogenetic bands to roughly equal extents. Thus, there is no gross bias in the cloning of DNA from different bands into large YACs. To test band assignments further, hybridizations were carried out blind, and band positions were then compared with (1) probe localizations in cases in which a reported location was present in one of the YACs; (2) cross-hybridization of a labeled YAC with others in the collection; and (3) hybridization to a panel of DNAs from a series of hybrid cells containing Xq DNA truncated at various regions. Of 31 cases in which YACs contained a probe with a previously reported location, 28 in situ assignments were in agreement, and 14 other assignments, including one of the three discordant with probe localization, were confirmed by YAC cross-hybridization studies. Results with a group of nine YACs were further confirmed with a panel of somatic cell hybrid DNAs from that region. Five YACs hybridized both to Xq25 and to a second site (four in Xq27 and one in Xq28), suggestive of some duplication of DNA of the hybrid cell and perhaps in normal X chromosomes. The in situ assignments are thus sufficient to place YACs easily and systematically within bins of about 7-10 Mb and to detect some possible anomalies. Furthermore, on the basis of expectations for random cloning of DNA in YACs, the assigned YACs probably cover more than 50% of the total Xq24-Xq28 region. This provides one way to initiate the assembly of YAC contigs over extended chromosomal regions.

Base Sequence↗