Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Influence of paternally imprinted genes on development.

The parental origin of chromosomes is critical for normal development in the mouse because some genes are imprinted resulting in a predetermined preferential expression of one of the alleles. Duplication of the paternal (AG: androgenones) or maternal (GG/PG: gynogenones/parthenogenones) genomes will result in an excess or deficiency of gene dosage with corresponding phenotypic effects. Here, we report on the effects of paternally imprinted genes on development following introduction of the AG inner cell mass into normal blastocysts. There was a striking increase in embryonic growth by up to 50%, and a characteristic change in embryonic shape, partly because of the corresponding increase in length of the anterior-posterior axis. These changes, between e12-e15, were proportional to the contribution from AG cells to the embryo. However, a contribution of AG cells in excess of 50% was invariably lethal as development progressed to e15. A limited number of chimeras were capable of full-term development provided there was a relatively low contribution from AG cells. The distribution of AG cells in chimeras was not uniform, especially later in development when there was a disproportionate presence of AG cells in the mesodermally derived tissues. Their contribution was consistently greater in the heart and skeletal muscle, but was considerably lower in the brain. Chimeras detected after birth were either dead or developed severe abnormalities of the skeletal elements, particularly of the ribs which were enlarged, distorted and fused, with greatly increased cartilaginous material with an absence of normal ossification. These phenotypic effects in chimeras are reciprocal to those observed in the presence of GG/PG cells, which resulted in a substantial size reduction approaching 50%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single channel kinetics of a glutamate receptor.

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.     

New and noteworthy South American species of Agalinis (Scrophulariaceae)

Agalinis bandeirensis is a Brazilian species distinguished by linear bracts, short pedicels, and villous stamens.Agalinis ramulifera, from southern Brazil, is distinguished by many, short, leafy branches and small flowers.Agalinis linarioides subsp.rojasi, from Paraguay, is distinguished by paniculate inflorescences, short calyx lobes, and broad corollas.Gerardia bangii, G. digitalis, andG. meyeniana are recognized as species ofAgalinis and appropriate transfers are made.     

Microtubule assembly kinetics. Changes with solution conditions.

The assembly kinetics of microtubule protein are altered by ionic strength, temperature and Mg2+, but not by pH. High ionic strength (I0.2), low temperature (T less than 30 degrees C) and elevated Mg2+ (greater than or equal to 1.2 mM) induce a transition from biphasic to monophasic kinetics. Comparison of the activation energy obtained for the fast biphasic step at low ionic strength (I0.069) shows excellent agreement with the values obtained at high ionic strength, low temperature and elevated Mg2+. From this observation it can be implied that the tubulin-containing reactant of the fast biphasic event is also the species that elongates microtubules during monophasic assembly. Second-order rate constants for biphasic assembly are 3.82(+/- 0.72) x 10(7) M-1.s-1 and 5.19(+/- 1.25) x 10(6) M-1.s-1, and for monophasic assembly the rate constant is 2.12(+/- 0.56) x 10(7) M-1.s-1. The microtubule number concentration is constant during elongation of microtubules for biphasic and monophasic assembly.     

The thymic microenvironment. Characterization of in vitro differentiation of the IT26R21 rat thymic epithelial cell line

We have previously postulated an in vivo pathway of thymic epithelial (TE) cell maturation in pre- and postnatal thymus, whereby endocrine medullary TE cells terminally differentiate to form Hassall's bodies. Epithelial-cell differentiation has been well documented in vitro using epidermal keratinocytes. Therefore, to characterize TE-cell differentiation in vitro, we observed clones of the rat TE cell line, IT26R21, after 4 and 14 days in culture. We found alterations in cell morphology, the cessation of cell proliferation, and the acquisition of a differentiation antigen defined by monoclonal antibody TE-19 (a marker of terminally differentiated epithelial cells). At light and electron microscopy, we detected progressive TE-cell stratification and squamous-cell formation between 4 and 14 days of culture. Autoradiography on day 14 showed that squamous TE cells in stratified layers did not incorporate tritiated thymidine, while surrounding smaller cells adhering to the substratum continued to synthesize DNA. At indirect immunofluorescence, only 3% of cells reacted with monoclonal antibody TE-19 at day 4, while on day 14, 22% of the TE cells were TE-19 positive (P less than 0.02). Antibody-TE-19 reactivity was limited to stratified, squamous TE cells. Additionally, we isolated a clone of the IT26R21 cell line that did not undergo these changes characteristic of TE cell differentiation. We conclude that IT26R21 TE cells are capable of undergoing programs of both terminal differentiation and cell renewal in vitro.