Transforming mutant v-mos protein kinases that are deficient in in vitro autophosphorylation.

We investigated the importance of specific serine residues for autophosphorylation and transformation by serine-threonine protein kinase p37mos. When either serine 326 or 358 was replaced with alanine, the resulting mutant protein retained the ability to transform NIH 3T3 cells but failed to autophosphorylate in vitro. These studies represent the first functional uncoupling of these two activities for p37mos.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia.

Achondroplasia, the most common genetic form of dwarfism, is an autosomal dominant disorder whose underlying mechanism is a defect in the maturation of the cartilage growth plate of long bones. Achondroplasia has recently been shown to result from a Gly to Arg substitution in the transmembrane domain of the fibroblast growth factor receptor 3 (FGFR3), although the molecular consequences of this mutation have not been investigated. By substituting the transmembrane domain of the Neu receptor tyrosine kinase with the transmembrane domains of wild-type and mutant FGFR3, the Arg380 mutation in FGFR3 is shown to activate both the kinase and transforming activities of this chimeric receptor. Residues with side chains capable of participating in hydrogen bond formation, including Glu, Asp, and to a lesser extent, Gln, His and Lys, were able to substitute for the activating Arg380 mutation. The Arg380 point mutation also causes ligand-independent stimulation of the tyrosine kinase activity of FGFR3 itself, and greatly increased constitutive levels of phosphotyrosine on the receptor. These results suggest that the molecular basis of achondroplasia is unregulated signal transduction through FGFR3, which may result in inappropriate cartilage growth plate differentiation and thus abnormal long bone development. Achondroplasia may be one of the number of cogenital disorders where constitutive activation of a member of the FGFR family leads to development abnormalities.     

MONOPHYLY OF ANEUPLOID ASTRAGALUS (FABACEAE): EVIDENCE FROM NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Evolutionary relationships within Astragalus L. (Fabaceae) were inferred from nucleotide sequence variation in nuclear ribosomal DNA of both New World and Old World species. The internal transcribed spacer regions (ITS) of 18S–26S nuclear ribosomal DNA from representatives of 26 species of Astragalus, three species of Oxytropis DC., and two outgroup taxa were analyzed by polymerase chain reaction amplification and direct DNA sequencing. The length of the ITS 1 region within these taxa varied from 221 to 231 bp, while ITS 2 varied in length from 207 to 217 bp. Of the aligned, unambiguous positions, approximately 34% were variable in each spacer region. In pairwise comparisons among Astragalus species and outgroup taxa, sequence divergence at these sites ranged from 0 to 18.8% in ITS 1 and from 0 to 21.7% in ITS 2. Parsimony analyses of these sequences resulted in a well-resolved phylogeny that is highly concordant with previous cytogenetic and chloroplast DNA evidence for a major phylogenetic division in the genus. These data suggest that the New World aneuploid species of Astragalus form a monophyletic but morphologically cryptic group derived from euploid species of Old World (Eurasian) origin, which are consequently paraphyletic.     

Phylogenetic relationships in Maloideae (Rosaceae): evidence from sequences of the internal transcribed spacers of nuclear ribosomal DNA and its congruence with morphology

We used sequences from both internal transcribed spacers (ITS) and a small portion of the 5.8S gene of nuclear ribosomal DNA (nrDNA) for phylogenetic reconstruction of 19 genera of Maloideae and four potential outgroups from the Rosaceae. Parsimony analyses indicate that Maloideae are not monophyletic; Vauquelinia, which is traditionally placed in Spiraeoideae, and two genera of the Maloideae, Eriobotrya and Rhaphiolepis, form a well-supported clade that is the sister to the remainder of the subfamily. Although our ITS phylogenetic hypothesis is highly resolved, there is considerable homoplasy, and support, as indicated by bootstrap values and decay indices, is relatively weak for all groups except four: Eriobotrya-Rhaphiolepis-Vauquelinia, Crataegus-Mespilus, Amelanchier-Peraphyllum-Malacomeles, and Cydonia-Pseudocydonia. Our DNA sequence data do not support a broad interpretation of Sorbus. Intergeneric hybridization, which is prevalent in Maloideae, occurs between genera that are far removed from one another on our most-parsimonious trees. We infer an overall phylogeny from separate analyses of ITS DNA sequences and recently published morphological and wood anatomical studies of Maloideae and from analyses after pooling these data sets. The four most strongly supported clades of the ITS phylogeny appear in the phylogeny based on pooled data.     

Construction of a hybrid bacteriophage-plasmid recombinant DNA vector.

A phage-plasmid hybrid was constructed for use as a recombinant DNA vector, allowing the propagation of cloned EcoRI restriction endonuclease fragments of about 2 X 10(6) to 11 X 10(6) daltons. The colicin E1 plasmid replicon was fused to the left arm of a lambdagt generalized transducing phage with a thermolabile repressor, yielding a genome which could be replicated either by phage lambda functions or via the colicin E1 plasmid replicon. At the nonpermissive temperature, phage functions were derepressed and phage growth occurred lytically. Alternatively, at the permissive temperature, lambda functions were repressed and the vector replicated as a covalently closed circular plasmid. The phage-plasmid hybrid vector could be maintained at a copy number determined by the colicin E1 plasmid replicon and was also sensitive to amplification after chloramphenicol treatment. An EcoRI fragment of Escherichia coli DNA encoding genes of the arabinose operon also was inserted into the central portion of the vector.