Use of Genetic and Physical Mapping to Locate the Spinal Muscular Atrophy Locus between Two New Highly Polymorphic DNA Markers

The gene for autosomal recessive forms of spinal muscular atrophy (SMA) has recently been mapped to chromosome 5ql3, within a 4-cM region between the blocks D5S465/D5S125 and MAP-1B/D5S112. We identified two new highly polymorphic microsatellite DNA markers—namely, AFM265wf5 (D5S629) and AFM281yh9 (D5S637)—which are the closest markers to the SMA locus. Multilocus analysis by the location-score method was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between locus D5S629 and the block D5S637/D5S351/MAP-1B/D5S112/D5S357. Genetic analysis of inbred SMA families, based on homozygosity by descent and physical mapping using mega-YACs, gave additional information for the loci order as follows: cen–D5S6–D5S125/D5S465–D5S435–D5S629–SMA–D5S637–D5S351–MAP–1B/D5S112–D5S357–D5S39–tel. These data give the direction for bidirectional walking in order to clone this interval and isolate the SMA gene.     

The GLN1 Locus of SACCHAROMYCES CEREVISIAE Encodes Glutamine Synthetase

Among 41 yeast glutamine auxotrophs, complementation analysis defined a single gene, GLN1, on chromosome 16 between MAK3 and MAK6. Half of the alleles fell into two intragenic complementation classes. No clustering of complementing alleles was found in a fine structure map. Altered glutamine synthetase subunits, including nonsense fragments and charge variants, were identified in several of the mutants, indicating that GLN1 is the structural gene for this enzyme. Negative complementation was observed for almost every allele associated with a protein product and all gln1/+ heterozygotes displayed reduced susceptibility to ammonia repression of the remaining glutamine synthetase activity. This latter observation is explained by the hypothesis that ammonia represses the enzyme only through its metabolism to glutamine. A basis for the two gln1 complementation classes is proposed.     

The New Rga Locus Encodes a Negative Regulator of Gibberellin Response in Arabidopsis Thaliana

We have identified a new locus involved in gibberellin (GA) signal transduction by screening for suppressors of the Arabidopsis thaliana GA biosynthetic mutant ga1-3. The locus is named RGA for repressor of ga1-3. Based on the recessive phenotype of the digenic rga/ga1-3 mutant, the wild-type gene product of RGA is probably a negative regulator of GA responses. Our screen for suppressors of ga1-3 identified 17 mutant alleles of RGA as well as 10 new mutant alleles at the previously identified SPY locus. The digenic (double homozygous) rga/ga1-3 mutants are able to partially repress several defects of ga1-3 including stem growth, leaf abaxial trichome initiation, flowering time, and apical dominance. The phenotype of the trigenic mutant (triple homozygous) rga/spy/ga1-3 shows that rga and spy have additive effects regulating flowering time, abaxial leaf trichome initiation and apical dominance. This trigenic mutant is similar to wild type with respect to each of these developmental events. Because rga/spy/ga1-3 is almost insensitive to GA for hypocotyl growth and its bolting stem is taller than the wild-type plant, the combined effects of the rga and spy mutations appear to allow GA-independent stem growth. Our studies indicate that RGA lies on a separate branch of the GA signal transduction pathway from SPY, which leads us to propose a modified model of the GA response pathway.     

A Drosophila Third Chromosome Minute Locus Encodes a Ribosomal Protein

Minutes (M) are a group of over 50 phenotypically similar Drosophila mutations widely believed to affect ribosomal protein genes. This report describes the characterization of the P element-induced M(3)95A(Plac92) mutation [allelic to M(3)95A]. This mutation can be reversed by the mobilization of the P element, demonstrating that the mutation is caused by insertion of this transposable element. The gene interrupted by insertion of the P element was cloned by use of inverse polymerase chain reaction. Nucleotide sequence analysis revealed a 70-75% identity to the human and rat ribosomal protein S3 genes, and to the Xenopus ribosomal protein S1a gene. At the amino acid level, the overall identity is &78% for all three species. This is only the second time that a Minute has been demonstrated to encode a ribosomal protein.     

Repeated,Long-Term Cycling of Putative Stem Cells between Niches in a Basal Chordate

1. Download : Download high-res image (644KB)
2. Download : Download full-size image

Highlights? Identification of a niche for germline and somatic stem cells in a basal chordate ? Stem cells repeatedly migrate between aged or damaged niches into developing niches     

Direct Visualization of the Highly Polymorphic RNU2 Locus in Proximity to the BRCA1 Gene

Although the breast cancer susceptibility gene BRCA1 is one of the most extensively characterized genetic loci, much less is known about its upstream variable number tandem repeat element, the RNU2 locus. RNU2 encodes the U2 small nuclear RNA, an essential splicing element, but this locus is missing from the human genome assembly due to the inherent difficulty in the assembly of repetitive sequences. To fill the gap between RNU2 and BRCA1, we have reconstructed the physical map of this region by re-examining genomic clone sequences of public databases, which allowed us to precisely localize the RNU2 array 124 kb telomeric to BRCA1. We measured by performing FISH analyses on combed DNA for the first time the exact number of repeats carried by each of the two alleles in 41 individuals and found a range of 6-82 copies and a level of heterozygosity of 98%. The precise localisation of the RNU2 locus in the genome reference assembly and the implementation of a new technical tool to study it will make the detailed exploration of this locus possible. This recently neglected macrosatellite could be valuable for evaluating the potential role of structural variations in disease due to its location next to a major cancer susceptibility gene.     

The ripX Locus of Bacillus subtilis Encodes a Site-Specific Recombinase Involved in Proper Chromosome Partitioning

The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers in B. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripX strains by the first division after the initiation of germination. The introduction of a recA mutation into ripX strains resulted in only slight modifications of the ripX phenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing a dif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.     

Entamoeba histolytica Encodes a Highly Divergent β-Tubulin

ABSTRACT. The microtubules of the amitochondrial parasite Entamoeba histolytica are atypical in certain respects. Consistent with this, we report that E. histolytica encodes the most divergent β -tubulin identified to date, with only 54% to 58% identity to β -tubulins from various species. A similarly divergent β -tubulin is encoded by the related Entamoeba invadens ; single gene copies appear to be present in both organisms. The Entamoeba sequences were compared with a database of 101 β -tubulins, including the highly divergent sequence from another amitochondrial protozoan, Trichomonas vaginalis. A total of 81 residues were universally conserved, and 76 residues varied only once. Correlations with previous studies indicate that microtubule function is altered when most, but not all, conserved residues are mutated.     

Major Histocompatibility Locus of Rhesus Monkeys (RhL-A)

TISSUE typing in rhesus monkeys is an essential prerequisite of organ and bone marrow transplantation in this and other laboratories. Since our first demonstrations in 1965 that leucocyte antigens are relevant for histocompatibility in primates¹, two leucocyte specificities of rhesus monkeys have been defined serologically² and since then the number of available iso-antisera has gradually increased. Although most of the reagents are not monospecific, the inheritance of the leucocyte “groups” suggests that they are controlled by genes segregating as single units in a Mendelian fashion³, but the data previously obtained were insufficient for a thorough genetic analysis.     

A Highly Polymorphic Plasma Protein Locus in Brown Trout (Salmo trutta L.) Populations from Portugal

Genetic polymorphism of an unidentified plasma protein (PX) is described for the first time in Salmo trutta (L.) by means of isoelectric focusing. The analysis of 414 individuals from different geographic origins in Portugal allowed the identification of nine alleles. Heterozygosity in natural populations is generally above 0.60, thus giving similar values to those reported for brown trout microsatellite loci. Substructuring of Portuguese brown trout is evident between northern and southern basins. Genetic affinities between the southernmost rivers and the hatchery stock were detected, suggesting the existence of recent stocking influences.     

Vascular Regeneration in a Basal Chordate Is Due to the Presence of Immobile,Bi-Functional Cells

The source of tissue turnover during homeostasis or following injury is usually due to proliferation of a small number of resident, lineage-restricted stem cells that have the ability to amplify and differentiate into mature cell types. We are studying vascular regeneration in a chordate model organism, Botryllus schlosseri, and have previously found that following surgical ablation of the extracorporeal vasculature, new tissue will regenerate in a VEGF-dependent process within 48 hrs. Here we use a novel vascular cell lineage tracing methodology to assess regeneration in parabiosed individuals and demonstrate that the source of regenerated vasculature is due to the proliferation of pre-existing vascular resident cells and not a mobile progenitor. We also show that these cells are bi-potential, and can reversibly adopt two fates, that of the newly forming vessels or the differentiated vascular tissue at the terminus of the vasculature, known as ampullae. In addition, we show that pre-existing vascular resident cells differentially express progenitor and differentiated cell markers including the Botryllus homologs of CD133, VEGFR-2, and Cadherin during the regenerative process.     

The GA2 Locus of Arabidopsis thaliana Encodes ent-Kaurene Synthase of Gibberellin Biosynthesis

The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf. Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to ent-kaurene, which is catalyzed by ent-kaurene synthase (KS). Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype. A genomic clone coding for KS, AtKS, was isolated from A. thaliana using CmKS cDNA as a heterologous probe. The corresponding A. thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein. The fusion protein showed enzymatic activity that converted [³H]copalyl diphosphate to [³H]ent-kaurene. The recombinant AtKS protein derived from the ga2–1 mutant is truncated by 14 kD at the C-terminal end and does not contain significant KS activity in vitro. Sequence analysis revealed that a C-2099 to T base substitution, which converts Gln-678 codon to a stop codon, is present in the AtKS cDNA from the ga2–1 mutant. Taken together, our results show that the GA2 locus encodes KS.     

The HumanHNRPDLocus Maps to 4q21 and Encodes a Highly Conserved Protein

The hnRNP D protein interacts with nucleic acids bothin vivoandin vitro.Like many other proteins that interact with RNA, it contains RBD (or “RRM”) domains and arg-gly-gly (RGG) motifs. We have examined the organization and localization of the human and murine genes that encode the hnRNP D protein. Comparison of the predicted sequences of the hnRNP D proteins in human and mouse shows that they are 96.9% identical (98.9% similar). This very high level of conservation suggests a critical function for hnRNP D. Sequence analysis of the humanHNRPDgene shows that the protein is encoded by eight exons and that two additional exons specify sequences in the 3′ UTR. Use of two of the coding exons is determined by alternative splicing of theHNRPDmRNA. The humanHNRPDgene maps to 4q21. The mouseHnrpdgene maps to the F region of chromosome 3, which is syntenic with the human 4q21 region.     

The Drosophila Maternal Effect Locus Deadhead Encodes a Thioredoxin Homolog Required for Female Meiosis and Early Embryonic Development

This study describes the identification, function and molecular characterization of deadhead, a Drosophila thioredoxin homolog. Although in vitro studies have shown that thioredoxin can posttranslationally regulate the activity of many different proteins, we find that this homolog is not essential for viability. The phenotypic analysis of two different mutations which eliminate function suggests that dhd is essential for female meiosis. The majority of eggs laid by females homozygous for null mutations are fertilized but fail to complete meiosis. A small number of escaper embryos initiate development and display a range of phenotypes suggesting functions in both preblastoderm mitosis and head development. Our analysis of deadhead's RNA expression pattern is consistent with its maternal effect function: the RNA is predominately expressed in the nurse cells of the ovary, is maternally deposited into the egg, but does not appear to be zygotically expressed during embryogenesis. Thus both our genetic and molecular data are consistent with a function during meiosis and preblastoderm mitosis. Whether the head defect indicates an additional function or is an indirect consequence of earlier defects remains to be determined.