GENETIC LEAKAGE AFTER ADAPTIVE AND NONADAPTIVE DIVERGENCE IN THE ENSATINA ESCHSCHOLTZII RING SPECIES

The salamander Ensatina eschscholtzii is an example of a ring species in which extant intermediate stages of terminal forms have a nearly continuous range, offering replicated interactions at several stages of divergence. We employ a greatly expanded allozyme database and individual-based analyses to separate the effects of divergence time and gene flow to evaluate how gradual divergence of populations around the ring contributes to the development of reproductive isolation. Despite the high degree of genetic ( D ≤ 0.39) and ecomorphological divergence observed in secondary contacts around the ring, reproductive isolation or rare hybridization is observed only at the terminus of the ring. Instead, in the secondary contacts sampled around the ring, hybrids are common and reproductively successful, enabling genetic leakage between parental genomes and the potential for genetic merger. Nevertheless, genetic admixture is geographically broad (<100 km) only in contacts between ecomorphologically similar populations (within subspecies). When divergence is accompanied by alternative patterns of adaptive divergence (between subspecies), zones of intergradation are narrower and affect populations only locally (>8 km). Diversification and consequent genetic interactions in Ensatina reveal a continuum between populations, ecological races, and species, where polytypic traits and high genetic differentiation are maintained without reproductive isolation.     

Thyroxine-binding globulin variant (TBG-Kumamoto): identification of a point mutation and genotype analysis of its family.

Thyroxine-binding globulin (TBG) is the major thyroid hormone transport protein. Several inherited TBG variants resulting in partial or complete TBG deficiencies have been shown to be caused by either one or two nucleotide substitutions, or one nucleotide deletion in the coding regions of the TBG gene. In this report, a Japanese female patient (proband) with hyperthyroid state, whose lower TBG levels did not return to normal under the euthyroid state after treatment was examined. Genomic DNA samples from the proband with thyroxine-binding globulin deficiency (termed TBG-Kumamoto) and her family were subjected to the polymerase chain reaction, and the generated DNA fragments were sequenced. A single nucleotide substitution in the codon for the amino acid 363 of native TBG molecule (CCT to CTT) was found, resulting in the replacement of proline by leucine. It was revealed that the proband was a heterozygote and her father was a hemizygote. The mutation was confirmed by the allele-specific amplification of genomic DNAs from the proband and her father using oligonucleotide primers of normal or mutant residues at the 3' position in the polymerase chain reaction. These results indicate that the abnormality of TBG-Kumamoto is the consequence of this mutation. Genetically, this point mutation observed in TBG-Kumamoto might be classified as a new type of TBG deficiency.     

Breakdown and quantitation of the forked termination of replication intermediate of Bacillus subtilis

Using a procedure that minimizes shear forces, the BamHI-derived forked termination of replication intermediate of Bacillus subtilis, called band I DNA, can be extracted with little or no accompanying band II DNA. It has been shown that band II DNA is a product of band I breakdown. Nuclease P1-mediated breakdown of the forked band I DNA proceeds in two steps. The first causes the release of one of the arms as band II DNA; in the second step, the remaining arm is cleaved away to yield the free stem. It is concluded that band I represents the primary termination of replication intermediate. A quantitative assessment of the level of band I in DNA from cells of the merodiploid strain, GSY1127, growing at different rates has been made. For cells grown in a minimal medium, at least, the experimentally measured level of band I is of the order (approx. 60%) of that predicted for a complete block to movement of the clockwise fork at the replication terminus, terC.     

Expression of neuron-specific enolase in the pineal organ of the domestic fowl during post-hatching development

Immunohistochemistry for neuron-specific enolase (NSE) revealed that NSE is localized in both a limited number of pinealocytes and intrinsic afferent neurons in the pineal organ of the domestic fowl. Furthermore, a computer-assisted three-dimensional imaging technique allowed to clarify the reverse distributional pattern of both elements: NSE-positive pinealocytes displayed a dense distribution especially in the vesicular portion of the gland, whereas NSE-immunoreactive nerve cells were mainly found in the pineal stalk. The number of NSE-positive intrinsic neurons in the pineal organ of chickens decreased rapidly after hatching, with a concentration of these elements in the basal portion (stalk) of the pineal organ. On the other hand, immunoreactive pinealocytes increased remarkably in the end-vesicle of the organ with age, followed by a gradual expansion toward the proximal portion. Thus, the spectacular increase in NSE-positive pinealocytes and the progressive reduction of reactive neurons occurred in parallel during the course of post-hatching development. NSE-immunoreactive pinealocytes displayed morphological characteristics of bipolar elements, endowed with an apical protrusion into the pineal lumen and a short basal process at younger stages, whereas multipolar types of NSE-positive pinealocytes were predominantly found in the adult domestic fowl. These results indicate that in the pineal organ of the domestic fowl (1) the ontogenetic expansion of NSE-immunoreactive pinealocytes is paralleled by a regressive afferent innervation, (2) the NSE-positive pinealocytes transform from a bipolar (columnar) type to a multipolar type during post-hatching development, and (3) these ontogenetic changes in the NSE-immunoreactivity and morphology of pinealocytes may reflect the development of a neurosecretory-like capacity of the organ.     

Location of the dihydroorotase domain within trifunctional hamster dihydroorotate synthetase

Mammalian dihydroorotase (DHOase, EC 3.5.2.3) is part of a trifunctional protein, dihydroorotate synthetase which catalyzes the first three reactions of de novo pyrimidine biosynthesis. We have subcloned a portion of the cDNA from the plasmid pCAD142 and obtained a nucleotide sequence which extends 2.1 kb in the 5' direction from the sequence encoding the aspartate transcarbamoylase (ATCase) domain at the 3'-end of the cDNA. The DHOase and ATCase domains have been purified from an elastase digest of the trifunctional protein and subjected to amino acid (aa) sequencing from their N termini. The sequence of the N-terminal 24 aa of the DHOase domain has been obtained and aligned with the cDNA sequence. The C-terminal residues of the DHOase domain have been identified as Leu followed by Val which, when taken with partial sequences of the CNBr fragments of this domain, defines the coding sequence of the active, globular DHOase domain released by proteolysis. Prediction of protein secondary structure from the deduced aa sequence showed that the DHOase domain (Mr 37,751) is separated from the C-terminal ATCase domain (Mr 34,323) by a bridging sequence (Mr 12,532) consisting of multiple beta-turns.