Hair interior defect in AKR/J mice.

BACKGROUND: All AKR/J mice have a subtle defect that involves malformation of the central portion of hair fibres that is best visualized under white and polarized light microscopy. AIMS: This study sought to characterize the clinical and ultrastructural features of the hair interior defect (HID) phenotype and to determine the chromosomal localization of the hid mutant gene locus. METHODS: White and polarized light microscopy combined with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the HID phenotype. Complementation testing and gene-linkage studies were performed to map the locus. RESULTS: Using SEM, the hair-fibre structure on the surface was found to be similar to hairs obtained from normal BALB/cByJ+/+and C57BL/6 J+/+mice. There were also no differences in sulphur content. TEM revealed degenerative changes in the medulla similar to that seen by light microscopy. This autosomal recessive mutation is called HID (locus symbol: hid). We mapped the hid locus to the distal end of mouse chromosome 1. No genes reported to cause skin or hair abnormalities are known to be within this interval except for the lamin B receptor (Lbr), which had been excluded previously as the cause of the hid phenotype in AKR/J mice. CONCLUSION: A potentially novel gene or known gene with a novel phenotype resides within this interval, which may shed light on human diseases with defects in the inner structure of the hair fibre.

Role of procollagen mRNA levels in controlling the rate of procollagen synthesis.

Two factors must be present for primary avian tendon cells to commit 50% of their total protein production to procollagen: ascorbate and high cell density. Scorbutic primary avian tendon cells at high cell density (greater than 4 X 10(4) cells per cm2) responded to the addition of ascorbate by a sixfold increase in the rate of procollagen synthesis. The kinetics were biphasic, showing a slow increase during the first 12 h followed by a more rapid rise to a maximum after 36 to 48 h. In contrast, after ascorbate addition, the level of accumulated cytoplasmic procollagen mRNA (alpha 2) showed a 12-h lag followed by a slow linear increase requiring 60 to 72 h to reach full induction. At all stages of the induction process, the relative increase in the rate of procollagen synthesis over the uninduced state exceeded the relative increase in the accumulation of procollagen mRNA. A similar delay in mRNA induction was observed when the cells were grown in an ascorbate-containing medium but the cell density was allowed to increase. In all cases, the rate of procollagen synthesis peaked approximately 24 h before the maximum accumulation of procollagen mRNA. The kinetics for the increase in procollagen synthesis are not, therefore, in agreement with the simple model that mRNA levels are the rate-limiting factor in the collagen pathway. We propose that the primary control point is at a later step. Further support for this idea comes from inhibitor studies, using alpha, alpha'-dipyridyl to block ascorbate action. In the presence of 0.3 mM alpha, alpha'-dipyridyl there was a specific two- to threefold decrease in procollagen production after 4 h, but this was unaccompanied by a drop in procollagen mRNA levels. Therefore, inhibitor studies give further support to the idea that primary action of ascorbate is to release a post-translational block.

Spx1, a novel X-linked homeobox gene expressed during spermatogenesis.

Spx1, a novel mouse homeobox gene, encodes a homeodomain characteristic of the paired-like class of homeobox genes and has been mapped to the distal end of the X chromosome. Northern blot hybridization of adult tissues detected high levels of a single Spx1 transcript in the testis. Further analysis by in situ hybridization revealed predominant Spx1 expression within the spermatogonia/preleptotene spermatocytes and round spermatids of spermatogenic stages IV-VII. These expression data suggest SPX1 may play a role in the regulation of spermatogenesis.

Advantages of gradient vs. 5-fluorouracil enrichment of stem cells for retroviral-mediated gene transfer.

Retrovirally mediated gene transfer into murine totipotent hematopoietic stem cells (THSC) may be more efficient when the donor stem cells are enriched. We have used a rapid, nontoxic density gradient separation of mouse marrow to enrich stem cells. By characterizing the cell types in various fractions of the gradient, we found the majority of the THSC, spleen colony forming stem cells (CFU-S), erythroid burst forming cells (BFU-E) and dividing cells were in the same fraction. The gradient enrichment technique was then compared with one requiring 5-fluorouracil (5-FU) treatment of donor mice prior to marrow harvest. Cells enriched by both methods were tested for their ability to mediate retroviral gene transfer into normal mice. Gradient enrichment provided only one third as many nucleated cells as 5-FU treatment from the same number of donors. During the subsequent 4-day in vitro exposure to the retrovirus and growth factors, however, the number of gradient enriched cells increased 1.6-fold while the number of 5-FU treated cells decreased 3-fold. In lethally irradiated recipients, there was no difference between gradient and 5-FU enriched donor cells in the proportion of cells that generated CFU-S nor in the percentage of CFU-S that were infected. Secondary hosts did show differences. Gradient-enriched cells maintained more survivors for up to 6 months posttransplantation and more of the survivors were positive for the retrovirus. It is clear that the gradient method provides a rapid means to enrich CFU-S and THSC without exposure to the toxic effects of 5-FU.

Cross-referencing radiation hybrid data to the recombination map: lessons from mouse chromosome 18.

We are building a framework map of known-order anchor markers between the mouse T31 radiation hybrid (RH) panel and the recombination map based on The Jackson Laboratory (TJL) interspecific backcross panels using the established genetic order to evaluate and strengthen the RH results. In making this map comparison, we have elucidated several problems inherent in RH mapping and minimized these by careful attention to data gathering and interpretation methods. We describe lessons and pitfalls of developing radiation hybrid maps, using the example of mouse Chromosome 18, for which we have built a framework map of microsatellite anchor loci spanning the entire chromosome at significant LOD with no gaps. Sixty-five D18Mit- simple sequence length polymorphism (SSLP) markers form a continuous linkage along the T31 RH Chromosome 18 (RH map length 1598 cR, genetic length 41 cM) with all LODs greater than 6. These markers are also placed on TJL interspecific backcrosses, and the order of the markers in the two systems is in complete agreement. We are continuing to cross-reference the RH data to TJL backcross data for the other mouse chromosomes to improve further the power of RH mapping and to integrate more precisely the extensive existing recombination mapping data for the mouse with the incoming radiation hybrid map data.

Regulation of the galactose pathway in Saccharomyces cerevisiae: induction of uridyl transferase mRNA and dependency on GAL4 gene function.

In Saccharomyces cerevisiae, utilization of galactose requires four inducible enzyme activities. Three of these activities (galactose-1-phosphate uridyl transferase, EC 2.7.7.10; uridine diphosphogalactose 4-epimerase, EC 5.1.3.2; and galactokinase, EC 2.7.1.6) are specified by three tightly linked genes (GAL7, GAL10, and GAL1, respectively) on chromosome II, whereas the fourth, galactose transport, is specified by a gene (GAL2) located on chromosome XII. Although classic genetic analysis has revealed both positive and negative regulatory genes that coordinately affect the appearance of all four enzyme activities, neither the basic events leading to the appearance of enzyme activities nor the roles of the regulatory genes have yet been determined. Regulation of inducible enzyme activity could be mediated by events related to transcription, translation, or enzyme activation. For the purpose of studying galactose pathway induction and its regulation, we have developed an immunoprecipitation assay that enables us to detect the GAL7 specified uridyl transferase polypeptide in yeast extracts and among the polypeptides synthesized in an RNA-dependent in vitro translation system. Use of this immunoprecipitation assay in conjunction with in vivo labeling experiments demonstrates the presence of [(3)H]leucine-labeled transferase in extracts prepared from cells grown in galactose but not from cells grown in glucose. This galactose-specific induction of transferase polypeptide is mediated by the de novo appearance of a functional mRNA species whose synthetic capacity is detectable by the combination of in vitro translation and immunoprecipitation. The appearance of functional transferase mRNA depends on wild-type expression of the positive regulatory gene, GAL4. Cells carrying a nonsense (amber) mutation in the GAL4 gene fail to produce the transferase mRNA, whereas a nonsense suppressor of the GAL4 amber mutant regains the galactose-specific mRNA response. Our results establish that the induction of the GAL7 specified uridyl transferase activity is mediated by de novo appearance of a functional mRNA and that this galactose-specific response is dependent on a wild-type GAL4 gene product.

The sucrase-isomaltase structural gene (Si-s) and a regulatory gene (Si-r) are closely linked to esterase-26 (Es-26) on mouse chromosome 3.

A cDNA clone of the rat sucrase-isomaltase (SI) structural gene detected two distinct patterns of DNA fragments on Southern blots of mouse DNA. Screening of 18 AKXL and 25 AKXD recombinant inbred (RI) strains revealed that all bands in each pattern co-segregated and there were no (0/43) recombinants with Es-26 on mouse Chromosome (Chr) 3. Since CBA/CaJ mice have approximately threefold less sucrase activity than other strains, we intercrossed them with SJL/J mice to map the previously identified SI regulatory gene, Si-r. Fifty-six mice from the F2 generation were assayed for sucrase activity, and the genotype of the murine SI structural gene locus, Si-s, was determined by Southern blot analysis. Nine animals (16%) were homozygous for the CBA/CaJ allele (C) and had an average sucrase activity (jejunum+ileum) of 1.51 mumoles/h/mg protein (SE = 0.067), 19 (34%) were homozygous for the SJL/J allele (S) and had an average sucrase activity of 5.95 mumoles/h/mg protein (SE = 0.267), and 28 (50%) were heterozygous (C/S) for Si-s with an average sucrase activity of 3.70 mumoles/h/mg protein (SE = 0.127). We conclude that Si-s and Si-r are closely linked on Chr 3.

Genetic mapping of 18S ribosomal RNA-related loci to mouse chromosomes 5, 6, 9, 12, 17, 18, 19, and X.

The organization of ribosomal RNA genes (rDNA) in the genome of the mouse varies significantly from one strain to another, but has been shown to follow the pattern of clusters of tandem repeats located at chromosome ends, often associated with cytological nucleolus organizer regions. The number of copies of the repeat unit at each locus also varies. A probe for the 18S ribosomal RNA sequence on Southern blots reveals both high copy number bands and fainter bands indicative of low repeat number. We have mapped a number of newly identified low-copy-number rDNA loci in C57BL/6J, in addition to placing some of the NOR-associated rDNA repeats on the Jackson interspecific backcross (BSS) map. We suggest that additional low-copy-number loci may remain to be mapped, and that the evolution of rDNA loci in the genome may include the proliferation of single copies by retroinsertion or other mechanisms.

Mutations preventing transpositions of yeast mating type alleles.

Homothallic strains of Saccharomyces cerevisiae can switch from one mating type to the other as often as every cell division. The conversion of mating type alleles (from MATa to MATalpha or vice versa) depends on other, unexpressed copies of a or alpha information that can be transposed to MAT. Previously, "inconvertible" mutations within MATalpha and MATa have been described that block the excision of the MAT allele. In this paper we describe two cis-acting mutations that also impair mating type switching and lie very near, but outside, the MAT locus. Both "stuck" mutations, stk1 and stk2, diminish the efficiency of converting MATa to MATalpha to less than 10% of normal. The stk1 mutation also slightly reduces conversion of MATalpha to MATa, whereas stk2 has no discernible effect. Unlike the inconvertible MATalpha-inc and MATa-inc mutations within MAT, the stk mutations are not replaced by wild-type sequences after the "stuck" cells occasionally switch to the opposite mating type. Because these mutations are not "healed" by mating type conversions, they must lie in sequences outside of the transposable mating type information. These results indicate that the efficient replacement of MAT alleles depends on sequences both within and adjacent to the MAT locus. Among subclones of homothallic stk MATa strains, approximately 2% show "illegal" transpositions of mating type genes. In these colonies the silent copy of alpha information at the HMLalpha locus has been converted to a, without any change of MATa or the silent a copy at HMRa. Such conversions of the unexpressed library genes are not found in wild-type homothallic strains that can switch mating type efficiently, but they are found in MATa-inc and MATalpha-inc strains. It appears that all of the cis-acting mutations within or adjacent to mating type result in these unusual switches of mating type information at HML and HMR.

Translation of the L-species dsRNA genome of the killer-associated virus-like particles of Saccharomyces cerevisiae.

Virus-like particles containing the L (P1)-species of double-stranded RNA (dsRNA) were isolated from Saccharomyces cerevisiae, and the translational activity of the virus-like particle-derived dsRNA was analyzed in the wheat germ cell-free system. Denaturation of the dsRNA immediately prior to in vitro translation resulted in the synthesis of one major and at least three minor polypeptides, whereas undenatured dsRNA, as expected, did not stimulate [35S]methionine incorporation into polypeptides, but actually slightly inhibited endogenous activity. The major in vitro translation product of the denatured L-dsRNA was shown to be identical with the major L-dsRNA containing virus-like particle capsid polypeptide on the basis of three criteria: co-electrophoresis on sodium dodecyl sulfate polyacrylamide gels, immunoprecipitation, and tryptic peptide analysis. We have therefore established that the L-dsRNA genome encodes the major virus-like particle capsid polypeptide. This result adds considerable support to the hypothesis that the L-dsRNA genome acts as a helper genome to the smaller (1.6 x 10(6) dalton) M-dsRNA genome in killer strains of yeast by providing the M-dsRNA containing virus-like particles with their major coat protein.

Molecular and genetic characterization of GABP beta.

This report outlines three observations relating to GABP beta, a polypeptide constituent of the heterotetrameric transcription factor GABP. Evidence is presented showing that the mouse genome encodes two highly related GABP beta polypeptides, designated GABP beta 1-1 and GABP beta 2-1. Genomic and cDNA copies of the newly defined Gabpb2 gene were cloned and characterized, providing the conceptually translated amino acid sequence of GABP beta 2-1. The genes encoding these two proteins, as well as GABP alpha, were mapped to three unlinked chromosomal loci. Although physically unlinked, the patterns of expression of the three genes were strikingly concordant. Finally, the molecular basis of GABP beta dimerization was resolved. Carboxy-terminal regions of the two GABP beta polypeptides, which mediate dimerization, bear highly related primary amino acid sequences. Both sequences are free of alpha-helix destabilizing residues and, when displayed on idealized alpha-helical projections, reveal marked amphipathy. Two observations indicate that these regions adopt an alpha-helical conformation and intertwine as coiled-coils. First, the dimer-forming region of GABP beta 2-1 can functionally replace the leucine zipper of a bZIP transcription factor. Second, a synthetic peptide corresponding to this region shows distinctive helical properties when examined by circular dichroism spectroscopy. Finally, evidence is presented showing that GABP beta 1-1 and GABP beta 2-1 can heterodimerize through this carboxy-terminal domain, but neither protein can heterodimerize via the dimer-forming region of the bZIP protein C/EBP beta.

Genomic organization and chromosome localization of the murine homeobox gene Pmx.

Homeobox genes are expressed in very specific temporal and spatial patterns and function as transcriptional regulators of developmental processes. The murine homeobox gene, Pmx (paired mesoderm homeobox), is expressed in a mesodermally restricted pattern in embryos and most abundantly in cardiac, skeletal, and smooth muscle tissues in adults. Previously, this murine gene was named K-2 and mHox, while the human homolog was named Phox1. In this report, the localization of Pmx has been determined by interspecific backcross analysis. The Pmx gene is located on Chromosome 1, approximately 3.3 cM distal to the Gsh-4 homeobox locus. The sequence of the Pmx transcript has been extended toward the 5' end and corresponds in size to one of the transcripts previously detected by Northern blot analysis. Sequence analysis indicates that Pmx is the first characterized mammalian gene to encode a paired type homeodomain, but not a paired domain. The Pmx gene includes at least five exons spanning a minimum of 60 kb of genomic DNA, making this the largest known murine homeobox gene.

High-resolution maps of the murine Chromosome 2 region containing the cholesterol gallstone locus, Lith1.

The Lith1 region on Chromosome (Chr) 2 contains a gene that markedly affects the prevalence of cholesterol gallstones in inbred mice. We report the high-resolution genetic and radiation hybrid maps of the chromosomal region surrounding Lith1, using three resources: a DNA panel from 188 progeny from two reciprocal backcrosses between C57BL/6 and Mus spretus inbred strains; 423 progeny of an N4 generation from backcrossing the susceptible C57L/J alleles at Lith1 into the resistant AKR/J strain; and the newly developed hamster-mouse T31 radiation hybrid panel. We mapped 17 microsatellite markers in the D2Mit182 to D2Mit14 region and two candidate genes for Lith1, the canalicular bile salt export pump (Bsep) also known as sister of P-glycoprotein (Spgp) and the low-density-lipoprotein-receptor-related gene megalin (Gp330). Both genetic maps were in agreement and ordered the microsatellite markers into a 10.4 +/- 1.5 cM region. The high-resolution physical map revealed ordering of microsatellite markers and relative distances between markers in almost complete agreement with the genetic maps. Mapping of Bsep revealed its location on Chr 2, homologous to the human chromosomal position (Nature Genet 20, 233-238, 1998). The radiation hybrid results also provided the highest resolution of the area containing the two candidate genes, which both mapped in the Lith1 region with close linkage, being separated by a distance of only 15 cR(3000). The total radiation hybrid map length of the region between D2Mit182 and D2Mit14 was 326 cR(3000), suggesting that 31 cR(3000) is equivalent to 1 cM in this region of Chr 2.

Identification and genetic mapping of a new polycystic kidney disease on mouse chromosome 8.

We report here a new mouse mutation, kat, that causes pleiotropic effects including facial dysmorphism, dwarfing, male sterility, anemia, and progressive polycystic kidney disease. kat (kidney anemia and testis) and a second allele, kat2J, that occurred on C57BL/ 6J were mapped to mouse chromosome (Chr) 8 using intra- and intersubspecific intercrosses. A high-resolution map for kat2J on Chr 8 was constructed using the F2 progeny from a cross between C57BL/6J-kat2J/+ and an inbred strain of Mus musculus castaneus (CAST/Ei). The kat2J mutation was localized between D8Mit129 and D8Mit128 with the gene order centromere-D8Mit100-(1.2 +/- 0.26 cM)-D8Mit231-(0.17 +/- 0.09 cM)-D8Mit129-(0.28 +/- 0.12 cM)-D8Mit128-(0.98 +/- 0.23 cM)-D8Mit25/D8Mit8. This segment is homologous to human Chr 19p. The two mutations at this locus that have occurred at The Jackson Laboratory will be invaluable for positional cloning and subsequent functional analysis of the mutated gene.

Reversal of pathology in murine mucopolysaccharidosis type VII by somatic cell gene transfer.

An inherited deficiency of beta-glucuronidase in humans, mice and dogs causes mucopolysaccharidosis VII (Sly syndrome), a progressive degenerative disease that reduces lifespan (to an average of 5 months in mice) and results from lysosomal storage of undegraded glycosaminoglycans in the spleen, liver, kidney, cornea, brain and skeletal system. Bone marrow transplantation in mutant mice provides a source of normal enzyme ('cross-correction'), which substantially improves the clinical condition and extends the average lifespan to 18 months. Gene therapy by transfer of a beta-glucuronidase gene into mutant haematopoietic stem cells is an alternative approach, but it is not known whether the low expression of vector-transferred genes in vivo would be sufficiently effective. Here we show that retroviral vector-mediated transfer of the gene to mutant stem cells results in long-term expression of low levels of beta-glucuronidase which partially corrects the disease by reducing lysosomal storage in liver and spleen.

Localization of the tight junction protein gene TJP1 to human chromosome 15q13, distal to the Prader-Willi/Angelman region, and to mouse chromosome 7.

The gene encoding the tight junction (zonula occludens) protein, TJP1, was mapped to human chromosome 15q13 by fluorescence in situ hybridization (FISH) using a cDNA probe. The Jackson Laboratory backcross DNA panel derived from the cross (C57BL/6JEi x SPRET/Ei) F1 females x SPRET/Ei males was used to map the mouse Tjp1 to chromosome 7 near position 30 on the Chromosome Committee Map, a region with conserved homology to human chromosome 15q13. FISH studies on metaphases from patients with the Prader-Willi (PWS) or the Angelman syndrome (AS) showed that TJP1 maps close but distal to the PWS/AS chromosome region.

Genetic mapping of 262 loci derived from expressed sequences in a murine interspecific cross using single-strand conformational polymorphism analysis.

We have demonstrated previously that noncoding sequences of genes are a robust source of polymorphisms between mouse species when tested using single-strand conformation polymorphism (SSCP) analysis, and that these polymorphisms are useful for genetic mapping. In this report we demonstrate that presumptive 3'-untranslated region sequence obtained from expressed sequence tags (ESTs) can be analyzed in a similar fashion, and we have used this approach to map 262 loci using an interspecific backcross. These results demonstrate SSCP analysis of genes or ESTs is a simple and efficient means for the genetic localization of transcribed sequences, and is furthermore an approach that is applicable to any system for which there is sufficient sequence polymorphism.

Maps from two interspecific backcross DNA panels available as a community genetic mapping resource.

We established two mouse interspecific backcross DNA panels, one containing 94 N2 animals from the cross (C57BL/6J x Mus spretus)F1 x C57BL/6J, and another from 94 N2 animals from the reciprocal backcross (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei. We prepared large quantities of DNA from most tissues of each animal to create a community resource of interspecific backcross DNA for use by laboratories interested in mapping loci in the mouse. Initial characterization of the genetic maps of both panels has been completed. We used MIT SSLP markers, proviral loci, and several other sequence-defined genes to anchor our maps to other published maps. The BSB panel map (from the backcross to C57BL/6J) contains 215 loci and is anchored by 45 SSLP and 32 gene sequence loci. The BSS panel map (from the backcross to SPRET/Ei) contains 451 loci and is anchored by 49 SSLP loci, 43 proviral loci, and 60 gene sequence loci. To obtain a high density of markers, we used motif-primed PCR to "fingerprint" the panel DNAs. We constructed two maps, each representing one of the two panels. All new loci can be located with a high degree of certainty on the maps at current marker density. Segregation patterns in these data reveal several examples of transmission ratio distortion and permit analysis of the distribution of crossovers on individual chromosomes.

A high-resolution radiation hybrid map of the proximal portion of mouse chromosome 5.

Radiation hybrid (RH) mapping of the mouse genome provides a useful tool in the integration of existing genetic and physical maps, as well as in the ongoing effort to generate a dense map of expressed sequence tags. To facilitate functional analysis of mouse Chromosome 5, we have constructed a high-resolution RH map spanning 75 cM of the chromosome. During the course of these studies, we have developed RHBase, an RH data management program that provides data storage and an interface to several RH mapping programs and databases. We have typed 95 markers on the T31 RH panel and generated an integrated map, pooling data from several sources. The integrated RH map ranges from the most proximal marker, D5Mit331 (Chromosome Committee offset, 3 cM), to D5Mit326, 74.5 cM distal on our genetic map (Chromosome Committee offset, 80 cM), and consists of 138 markers, including 89 simple sequence length polymorphic markers, 11 sequence-tagged sites generated from BAC end sequence, and 38 gene loci, and represents average coverage of approximately one locus per 0.5 cM with some regions more densely mapped. In addition to the RH mapping of markers and genes previously localized on mouse Chromosome 5, this RH map places the alpha-4 GABA(A) receptor subunit gene (Gabra4) in the central portion of the chromosome, in the vicinity of the cluster of three other GABA(A) receptor subunit genes (Gabrg1-Gabra2-Gabrb1). Our mapping effort has also defined a new cluster of four genes in the semaphorin gene family (Sema3a, Sema3c, Sema3d, and Sema3e) and the Wolfram syndrome gene (Wfs1) in this region of the chromosome.