Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters.

A triplex PCR method was developed to simultaneously amplify a heat-labile toxin sequence (LT) of 258 bp, a shiga-like toxin I sequence (SLT I) of 130 bp, and a shiga-like toxin II sequence (SLT II) of 346 bp from toxigenic strains of Escherichia coli. This method was used to screen 377 environmental E. coli isolates from marine waters or estuaries located in Southern California and North Carolina for enterotoxigenic or enterohemorrhagic E. coli strains. Of the 377 E. coli screened, one isolate was found to belong to the enterotoxigenic group, since it contained a LT homologous sequence, and one isolate was found to belong to the enterohemorrhagic group, since it contained a SLT I homologous sequence. None was found to contain SLT II homologous sequences. The pathogenicity of the positive environmental E. coli isolates was confirmed by standard bioassays with Y-1 adrenal cells and Vero cells to confirm toxin production. Our results suggest that toxigenic E. coli occurs infrequently in environmental waters and that there is a low public health risk from toxigenic E. coli in coastal waters.     

Deletion mapping of the rotavirus metalloprotein NS53 (NSP1): the conserved cysteine-rich region is essential for virus-specific RNA binding.

NS53 (NSP1), the gene 5 product of the group A rotaviruses, is a minor nonstructural protein of 486 to 495 amino acids which binds zinc and contains an amino-terminal highly conserved cysteine-rich region that may form one or two zinc fingers. To study the structure-function of the gene 5 product, wild-type and mutant forms of NS53 were produced by using a recombinant baculovirus expression system and a recombinant vaccinia virus/T7 (vTF7-3) expression system. Analysis of the RNA-binding activity of the wild-type NS53 immobilized onto protein A-Sepharose beads with NS53-specific antiserum showed that the protein exhibited specific affinity for all 11 rotavirus mRNAs. The use of short virus-specific RNA probes indicated that NS53 specifically recognizes an element located near the 5' ends of viral mRNAs. Analysis of the RNA-binding activity of deletion mutants of NS53 showed that the RNA-binding domain resides within the first 81 amino acids of the protein and that the highly conserved cysteine-rich region within this region of the protein is essential for the activity. Gel electrophoresis and Western immunoblot analyses of intracellular fractions derived from infected cells revealed that large amounts of NS53 were present in the cytosol and in association with the cytoskeletal matrix. Indirect immunofluorescence analysis of cells programmed to transiently express mutant forms of NS53 using vTF7-3 indicated that the intracellular localization domain resides between amino acids 84 and 176 of NS53. Together, these data show that the RNA-binding domain and the intracellular localization domain lie upstream from the region of NS53 previously determined not to be essential for replication of rotaviruses in cell culture (J. Hua and J. T. Patton, Virology 198:567-576, 1994).     

Serologic response of the opossum Didelphis virginiana to a temperature-sensitive mutant (ts-4) of Toxoplasma gondii.

A study was designed to measure the Toxoplasma gondii-specific IgM and IgG antibody responses of opossums inoculated with tachyzoites of the temperature-sensitive mutant of T. gondii, ts-4, and to examine its persistence in the tissues. Four young opossums seronegative for anti-Toxoplasma gondii IgM and IgG antibodies immediately after capture and 4 wk later were injected subcutaneously with 1.8 x 10(6) ts-4 tachyzoites; a fifth opossum (also seronegative) received an injection of saline only. Serum was collected weekly and titered by modified direct agglutination for anti-Toxoplasma gondii IgM and IgG. IgM titers were detectable from week 1 to week 6 postinoculation (PI). IgG was measurable by week 3 and remained high for 30 wk PI when the opossums were killed and examined. The control opossum did not develop a specific antibody response. At necropsy major lesions were not found. No anti-Toxoplasma gondii IgG was detected in serum collected from mice injected with tissues prepared from the opossums at necropsy, and no T. gondii was found on impression smears made at necropsy from these mice. Modified direct agglutination performed with or without 0.2 M 2-mercaptoethanol worked well for measuring specific IgM and IgG antibodies in experimentally infected opossums.     

Relationships among didelphid marsupials based on sequence variation in the mitochondrial cytochrome B gene

Variation in the mitochondrial cytochrome b gene (nucleotide and amino acid sequences) is evaluated for 9 genera and 15 species of American opossums in the family Didelphidae, using the American caenolestid rat opossumLestoros and the New Guinean peroryctid bandicootEchimypera as outgroups. Phylogenetic analyses (parsimony and distance) strongly support the monophyly of the Didelphidae and delineate two major clades; (1)Didelphis andPhilander are strongly aligned sister taxa, withMetachirus weakly but consistently associated with them, and (2)Marmosa plusMicoureus, withMonodelphis falling outside that pair. The generaMarmosops, Caluromys, andGlironia exhibit varied relationships, depending upon the method of analysis and data (DNA or amino acid sequences) used, but generally are placed individually or in combinations near or at the base of the didelphid radiation. Some aspects of these relationships are consistent with current taxonomic views, but others are in marked contrast. Specifically, a clade comprised of the mouse opossumsMarmosa, Micoureus, andMarmosops is strongly rejected by log-likelihood analysis, contrary to expectations from some current classifications. Also, the woolly opossumsCaluromys andGlironia also do not form a sister-taxon relationship, as suggested by their placement in a subfamily separate from the remaining didelphids examined. However, such a relationship cannot be rejected from log-likelihood analyses. The relationships suggested fromcyt-b sequences are strongly concordant with those based on DNA-DNA hybridization analyses. In addition to systematic and phylogenetic properties, molecular evolution of the didelphid cytochrome b gene sequence is characterized according to nucleotide bias and rate differentials at each codon position and across the entire sequence.To whom correspondence should be addressed.     

Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome.

Rotavirus cores contain the double-stranded RNA (dsRNA) genome, RNA polymerase VP1, and guanylyltransferase VP3 and are enclosed within a lattice formed by the RNA-binding protein VP2. Analysis of baculovirus-expressed core-like particles (CLPs) has shown that VP1 and VP2 assemble into the simplest core-like structures with replicase activity and that VP1, but not VP3, is essential for replicase activity. To further define the role of VP1 and VP2 in the synthesis of dsRNA from viral mRNA, recombinant baculoviruses containing gene 1 (rBVg1) and gene 2 (rBVg2) of SA11 rotavirus were generated and used to express recombinant VP1 (rVP1) and rVP2, respectively. After purification, the proteins were assayed individually and together for the ability to catalyze the synthesis of dsRNA in a cell-free replication system. The results showed that dsRNA was synthesized only in assays containing rVP1 and rVP2, thus establishing that both proteins are essential for replicase activity. Even in assays containing a primer-linked mRNA template, neither rVP1 nor rVP2 alone directed RNA synthesis. Characterization of the cis-acting replication signals in mRNA recognized by the replicase of rVP1 and rVP2 showed that they were the same as those recognized by the replicase of virion-derived cores, thus excluding a role for VP3 in recognition of the mRNA template by the replicase. Analysis of RNA-protein interactions indicated that the mRNA template binds strongly to VP2 in replicase assays but that the majority of the dsRNA product neither is packaged nor stably associates with VP2. The results of replicase assays performed with mutant VP2 containing a deletion in its RNA-binding domain suggests that the essential role for VP2 in replication is linked to the protein's ability to bind the mRNA template for minus-strand synthesis.     

Fine structural localization of potassium-stimulated rho nitrophenylphosphatase activity in denrites of the cerebral cortex

A histochemical technique for the demonstration of K+-rho-nitrophenylphosphatase (K+-rhoNPPase) activity, a component of the Na+,K+-ATPase, has been applied at the fine structural level in the somatosensory cortex of the rat. Reaction product was consistenly found in dendrites and in association with the cytoplasmic aspect of the dendritic plasmalemma. Reaction product often filled portions of the tubular smooth endoplasmic reticulum in these processes. The results of these studies are interpreted to indicate that enzymatic activity is associated with large-and small-diameter dendrites. No convincing evidence of high activity was found in glial profiles. The importance of neurons and their dendrites in active transport of sodium and potassium ions in the cerebral cortex may be more significant than indicated by studies with isolated neurons and glia.     

A method for isolation of a large amount of a single-stranded DNA fragment

Single-stranded DNA inserts can be digested from recombinant phage DNA of M13mp7 with BamHI or EcoRI restriction endonucleases. The single-stranded DNA is satisfactory for DNA sequencing and nuclei acid hybridization.     

Evolutionary genetics of birds. V. Genetic distances within Mimidae (mimic thrushes) and Vireonidae (vireos)

Genetic distances (D's) between five species within each of the families Mimidae and Vireonidae were estimated from frequencies of protein electromorphs at 23 loci. For three mimid species in the genus Toxostoma, equals 0.084 (range, 0.069–0.104); and among three mimid genera, equals 0.223 (0.167–0.278). These distances typify values previously reported in other birds at comparable levels of taxonomic recognition. In sharp contrast, the mean genetic distance among five congeneric species of Vireonidae is far higher, =0.360 (0.027–0.578). One possible explanation for these results is that Vireo species are considerably older, on the average, than are species of Toxostoma or than are members of several other avian genera assayed to date. Conventional thought about the origin and relative age of the Vireonidae appears compatible with this explanation. Although genetic distances in the Vireonidae are large by avian standards, they remain modest or even small in comparison with distances between many nonavian vertebrate congeners. Results for the Mimidae and the Vireonidae are directly contrasted with genetic distances in well-known genera of Amphibia and Reptilia.This research was supported by NSF Grant DEB 7814195 and by a grant from the American Philosophical Society.