Lisa Stubbs

Molecular characterization of radiation- and chemically induced mutations associated with neuromuscular tremors, runting, juvenile lethality, and sperm defects in jdf2 mice.

Abstract. The juvenile development and fertility-2 (jdf2) locus, also called runty-jerky-sterile (rjs), was originally identified through complementation studies of radiation-induced p-locus mutations. Studies with a series of ethylnitrosourea (ENU)-induced jdf2 alleles later indicated that the pleiotropic effects of these mutations were probably caused by disruption of a single gene. Recent work has demonstrated that the jdf2 phenotype is associated with deletions and point mutations in Herc2, a gene encoding an exceptionally large guanine nucleotide exchange factor protein thought to play a role in vesicular trafficking. Here we describe the molecular characterization of a collection of radiation- and chemically induced jdf2/Herc2 alleles. Ten of the 13 radiation-induced jdf2 alleles we studied are deletions that remove specific portions of the Herc2 coding sequence; DNA rearrangements were also detected in two additional mutations. Our studies also revealed that Herc2 transcripts are rearranged, not expressed, or are present in significantly altered quantities in animals carrying most of the jdf2 mutations we analyzed, including six independent ENU-induced alleles. These data provide new molecular clues regarding the wide range of jdf2 and p phenotypes that are expressed by this collection of recently generated and classical p-region mutations.

Molecular cloning of a novel mouse gene with predominant muscle and neural expression

Because numerous diseases affect the muscle and nervous systems, it is important to identify and characterize genes that may play functional roles in these tissues. Sequence analysis of a 106-kb region of human Chromosome (Chr) 19ql3.2 revealed a novel gene with homology to the Neuroendocrine-specific protein (NSP), and it has, therefore, been designated NSP-like 1 (Nspl1). We isolated the mouse homolog of this gene and performed extensive expression analysis of both the mouse and human genes. The mouse Nspl1 gene is alternatively spliced to produce two major transcripts: a 2.1-kb mRNA that is expressed at highest levels in the brain, and a 1.2-kb transcript that is primarily expressed in muscle. The larger message contains 10 exons, whereas the smaller transcript contains 7 exons. The last 6 exons, which are present in both transcripts, share significant amino acid sequence identity with the endoplasmic reticulum-bound portion of NSP. Mouse and human Nspl1/NSPL1 genes have expression patterns that are similar to that of the dystrophin gene. In addition, the putative regulatory domains of Nspl1 appear similar in composition and distribution to the defined dystrophin regulatory sequences.

Tandem Zinc-Finger Gene Families in Mammals: Insights and Unanswered Questions

Evidence for the remarkable conservation of mammalian genomes, in both content and organization of resident genes, is rapidly emerging from comparative mapping studies. The frequent occurrence of familial gene clustering, presumably reflecting a history of tandem in situ duplications starting from a single ancestral gene, is also apparent from these analyses. Genes encoding Kruppel-type zinc-finger (ZNF) proteins, including those containing Kruppel-associated box (KRAB) motifs, are particularly prone to such clustered organization. Existing data suggest that genes in KRAB-ZNF gene clusters have diverged in sequence and expression patterns, possibly yielding families of proteins with distinct, yet related, functions. Comparative mapping studies indicate that at least some of the genes within these clusters in mammals were elaborated prior to the divergence of mammalian orders and, subsequently, have been conserved. These data suggest a possible role for these tandem KRAB-ZNF gene families in mammalian evolution.

LOCATION OF MOUSE AND HUMAN GENES CORRESPONDING TO CONSERVED CANINE OLFACTORY RECEPTOR GENE SUBFAMILIES

Olfactory receptors are G protein-coupled, seven-transmembrane-domain proteins that are responsible for binding odorants in the nasal epithelium. They are encoded by a large gene family, members of which are organized in several clusters scattered throughout the genomes of mammalian species. Here we describe the mapping of mouse sequences corresponding to four conserved olfactory receptor genes, each representing separate, recently identified canine gene subfamilies. Three of the four canine genes detected related gene clusters in regions of mouse Chromosomes (Chrs) 2, 9, and 10, near previously mapped mouse olfactory genes, while one detected a formerly unidentified gene cluster located on mouse Chr 6. In addition, we have localized two human gene clusters with homology to the canine gene, CfOLF4, within the established physical map of Chr 19p. Combined with recently published studies, these data link the four conserved olfactory gene subfamilies to homologous regions of the human, dog, and mouse genomes.

The gene encoding the central cannabinoid receptor is located in proximal mouse Chromosome 4

Fig. 1. Genetic map location of Fxr on mouse Chr 10. Recombination fractions are given for adjacent loci with the first fraction representing data from the M. m. musculus crosses and the second fraction representing data from the M. spretus crosses. Gadl-psl was typed only in the M. spretus crosses, Matk only in the M. m. musculus crosses. Recombinational distances and standard errors are given in parenthesis. To the left of the map are the human map locations for the underlined genes.

Long-range walking techniques in positional cloning strategies

ConclusionThe past several years have seen an explosive growth in our understanding of the organization and structure of mammalian genomes, and refinements of existing techniques for genetic analysis, physical mapping, and large-fragment cloning techniques may well be enough to continue the momentum of that explosion for some time to come. Although refinement of existing techniques will certainly be necessary, the development of new and better cloning techniques may, perhaps, no longer be our most urgent need. The most important challenge that we face at present may in fact be that of finding efficient ways to share existing resources and information rapidly and equitably throughout the scientific community so that progress can continue unimpeded, and to catalog, correlate, and interpret the wealth of new data that is so rapidly accumulating.New strategies aimed at whole-genome mapping (Coulson et al. 1986, 1988; Michiels et al. 1987; Brenner and Livak 1989; Carrano et al. 1989; Lehrach et al. 1991) and sequencing (Church and Keifer-Higgins 1988; Bains and Smith 1988; Drmanac et al. 1989; Strzoska et al. 1991) may someday make the current method of long-range walking and physical mapping nearly passe. For example, since most of the relatively small nematode genome is now stored as ordered sets of cosmid and YAC clones (Coulson et al. 1986, 1988), a “walk” between a mapped marker and an uncloned gene can be accomplished rapidly, through a request for the appropriate series of clones from the ordered library. Vigorous drives by many laboratories to produce ordered clone libraries for murine and human chromosomes (Lehrach et al. 1991) may transform the process of cloning mammalian genes into a relatively trivial matter within the foreseeable future. The remarkable number of positional-cloning successes that have been reported in recent years may indicate that most of the best-defined, simply inherited mouse mutations and human hereditary disorders will have already been cloned by that time. When that is accomplished, the true challenging task will just begin: we must learn to decipher the complex biological programs encoded by our large and ever-growing storehouse of cloned, mapped and sequenced genes, before we can begin to understand what might be held in the vast “silent” mass of mammalian genomes.

The α2(XI) collagen gene lies within 8 kb of Pb in the proximal portion of the murine major histocompatibility complex

A number of serious hereditary disorders are now known to be associated with defective expression of collagen genes, and these findings have underscored the important and varied roles that the collagen family of genes must play during normal mammalian development. Although the activities of genes encoding the quantitatively major types of collagen are fairly well characterized, functions of the many minor types of collagen remain a matter of speculation. As a first step toward a functional analysis of type XI collagen, a member of this class of poorly understand “minor” collagen proteins which is expressed primarily in hyaline cartilage, we have used human probes for the gene encoding the proteins α2-subunit (COL11A2) to isolate and map homologous murine DNA sequences. Our results demonstrate that Col11a-2 is embedded within the major histocompatibility complex (MHC), within 8.4 kb of the class II pseudogene locus, Pb, and confirm that human and murine α2(XI) collagen genes are located in very similar genomic environments. The conserved location of these genes raises the possibility that type XI collagen genes may contribute to one or more of the diverse hereditary disorders known to be linked to the MHC in mouse and human.

The gene encoding adenylyl cyclase VII is located in central mouse Chromosome 8

Species: Mouse Locus name: Adenylyl cyclase 7 Locus symbol: Adcy7 Map position: Ucp, Junb-(2.56 +_ 1.46)-Adcy7-(7.69 +_ 2.46)Mr1; mouse Chromosome (Chr) 8. Method of mapping: I n t e r s p e c i f i c b a c k c r o s s [ ( C 3 H f / R MgfSl-2ZNU~g/+ X M. spretus) x C3Hf/R]; N = 117 (Stubbs et al., unpublished). Database deposit information: G e n b a n k access ion numbers D25538 (human) and U12919 (mouse). MGD-CREX-514 (mapping accession number) Molecular reagents: Probes used to map genes followed in this study included four cDNA sequences: Ucp (Ucp) [1]; 465.20 (Junb) [2];

The gene encoding sepiapterin reductase is located in central mouse Chromosome 6

49 (Adcy7) [3]; and mMt-1 (Mr1) [4]. The Junb and Mtl cDNA clones were obtained from the American Type Culture Collection (Rockville, Md.). Allele detection: DNA of backcross progeny were digested, transferred to Southern blots, and hybridized, as described [5]. Speciesspecific alleles used to follow each locus, and enzymes used to generate them, included: (1) Ucp: C3Hf/R1 (M) = 5.2 kb, M. spretus (S) = 0.5 kb, generated with PvulI; (2) Junb: M = 3.5 kb, S = 4.5 kb, Taql; (3) Adcy7: M = 16.2 kb, S = 8.5 kb, BamH1; (4) Mtl: M = 2.2 kb, S = 3.0 kb, Taql. Map positions were established by standard methods [6] with aid from the Map Manager data analysis program [7]. Published homologs: The human and mouse genes encoding adenylyl cyclase VII have been isolated (human: [3,8]; mouse: [9]). Discussion: Adenylyl cyclases, the enzymes responsible for convert ing ATP to cyclic AMP, play important roles in G-proteinregulated signal transduction systems. Chromosomal locations of the human and murine type I -VI adenylyl cyclases have recently been reported [10]. Hellevuo and associates [3] isolated a message for a novel adenylyl cyclase type (ADCY7) in the human erythroleukemia cell line HEL, which appears to be the major form of adenylyl cyclase in human platelets. ADCY7 has been mapped to human Chr 16q12-16q13 [11]. Interspecific backcross data presented here clearly establish l inkage between Adcy7 and several other genes located on mouse Chr 8 (Fig. I), with map positions

Location of the DBP transcription factor gene in human and mouse

References 1. Stanier P, Henson JN, Eddleston J, Moore GE, Copp AJ (1995) Genomics 26, 473-478 2. Krumlauf R, Holland PW, McVey JH, Hogan BLM (1987) Development 99, 603-617 3. Dietrich WF, Miller JC, Steen RG, Merchant M, Darrtron D, Nahf R, Gross A, Joyce DC, Wessel M, Dredge RD, Marquis A, Stein LD, Goodman N, Page DC, Lander ES (1994) Nature Genet 7, 220-245 4. Karn T, Holtrich U, Br~tuninger A, BtJhme B, Wolf G, RiibsamenWaidmann H, Strebhardt K (1993) Oncogene 8, 3433-3440 5. Lai C, Lemke G (1991) Neuron 6, 691-704 6. Lai C, Lemke G (1994) Oncogene 9, 877-883 7. Zerlin M, Julius MA, Goldfarb M (1993) Oncogene 8, 2731-2739 8. Johnson JD, Edman JC, Rutter WJ (1993) Proc Nail Acad Sci USA 90, 5677-5681 9. Perez JL, Shen X, Finkernagel S, Sciorra L, Jenkins NA, Gilbert DJ, Copeland NG, Wong TW (1994) Oncogene 9, 211-219 10. Edelhoff, S, Lai C, Disteche DM (1995) Genomics 25, 337-339 11. Lyon MF (1961) Genet Res 2, 92-95 12. Washburn L, Eicher EM (1986) Mouse News Lett 75, 28-29 13. Seldin MF (1996) Mamm Genome 6 (Suppl),