Evidence that the phytochrome gene family in black cottonwood has one PHYA locus and two PHYB loci but lacks members of the PHYC/F and PHYE subfamilies

The phytochrome photoreceptors play important roles in the photoperiodic control of vegetative bud set, growth cessation, dormancy induction, and cold-hardiness in trees. Interestingly, ecotypic differences in photoperiodic responses are observed in many temperate- zone tree species. Northern and southern ecotypes of black cottonwood (Populus trichocarpa Torr. & Gray), for example, exhibit marked differences in the timing of short-day-induced bud set and growth cessation, and these responses are controlled by phytochrome. Therefore, as a first step toward determining the molecular genetic basis of photoperiodic ecotypes in trees, we characterized the phytochrome gene (PHY) family in black cottonwood. We recovered fragments of one PHYA and two PHYB using PCR-based cloning and by screening a genomic library. Results from Southern analyses confirmed that black cottonwood has one PHYA locus and two PHYB loci, which we arbitrarily designated PHYB1 and PHYB2. Phylogenetic analyses which included PHY from black cottonwood, Arabidopsis thaliana and tomato (Solanum lycopersicum) suggest that the PHYB/D duplications in these species occurred independently. When Southern blots were probed with PHYC, PHYE, and PHYE heterologous probes, the strongest bands that we detected were those of black cottonwood PHYA and/or PHYB. These results suggest that black cottonwood lacks members of the PHYC/F and PHYE subfamilies. Although black cottonwood could contain additional PHY that are distantly related to known angiosperm PHY, our results imply that the PHY family of black cottonwood is less complex than that of other well-characterized dicot species such as Arabidopsis and tomato. Based on Southern analyses of five black cottonwood genotypes representing three photoperiodic ecotypes, substantial polymorphism was detected for at least one of the PHYB loci but not for the PHYA locus. The novel character of the PHY family in black cottonwood, as well as the differences in polymorphism we observed between the PHYA and PHYB subfamilies, indicates that a number of fundamental macro- and microevolutionary questions remain to be answered about the PHY family in dicots.      

Differences in human laminin B2 sequences.

A cDNA clone encoding the B2 chain of laminin has been isolated from a human endothelial lambda gt11 cDNA library. The nucleotide and deduced amino acid sequences of the clone were determined and showed one amino acid substitution (Ser1519 instead of Asn) when compared to lung laminin B2 chain and one silent nucleotide change (G4200 instead of A) in relation to the human placenta laminin B2 chain. Other differences in the 3'-untranslated region were also found.     

Testing the covarion hypothesis of molecular evolution

The covarion hypothesis of molecular evolution states that the fixation of mutations may alter the probability that any given position will fix the next change. Tests of this hypothesis using the divergence of real sequences are compromised because models of rate variation among sites (e.g., the gamma version of the one-parameter equation) predict sequence divergence values similar to those for the covarion process. This study therefore focuses on the extent to which the varied and unvaried codons of two well-diverged taxa are the same, because fewer are expected by the covarion hypothesis than by the gamma model. The data for these tests are the protein sequences of Cu, Zn superoxide dismutase (SOD) for mammals and plants. Simulation analyses show that the covarion hypothesis makes better predictions about the frequencies of varied and unhit positions in common between these two taxa than does the gamma version of the one-parameter model. Furthermore, the analysis of SOD tertiary structure demonstrates that mammal and plant variabilities are distributed differently on the protein. These results support the conclusions that the variable and invariable codons of mammal and plant SODs are different and that the covarion model explains the evolution of this protein better than the gamma version of the one-parameter process. Unlike other models, the covarion hypothesis accounts for rate fluctuations among positions over time, which is an important parameter of molecular evolution.      

Sterigmatocystin — incidence, fate and production byA versicolor in ras cheese

Sterigmatocystin (STG) is a toxic metabolite produced by severalAspergillus species. Because of its toxic and carcinogenic properties the occurrence of STG in food is considered to represent a potential hazard to man. The present study was designed to investigate following points:

Laminin-induced PC-12 cell differentiation is inhibited following laser inactivation of cellular prion protein

Prions, the etiological agents for infectious degenerative encephalopathies, act by inducing structural modifications in the cellular prion protein (PrPc). Recently, we demonstrated that PrPc binds laminin (LN) and that this interaction is important for the neuritogenesis of cultured hippocampal neurons. Here we have used the PC-12 cell model to explore the biological role of LN-PrPc interaction. Antibodies against PrPc inhibit cell adhesion to LN-coated culture plaques. Furthermore, chromophore-assisted laser inactivation of cell surface PrPc perturbs LN-induced differentiation and promotes retraction of mature neurites. These results point out to the importance of PrPc as a cell surface ligand for LN.     

ALKALINE RIBONUCLEASE ACTIVITY INCREASE IN RAT KIDNEY CORTEX AND LIVER AFTER TRYPAN BLUE AND OTHER AZO DYES ADMINISTRATION

Acid azo dyes, most of them naphtholdisulfonic acid derivatives, were given intraperitoneally to rats and their effect on "alkaline" ribonuclease activity was studied in total homogenates of kidney cortex and liver. Acid treatment was used to release bound enzyme activity. Several of the dyes, including trypan blue, increased RNase activity in both organs 3 days after administration of single doses, while others, like Evans blue, were inactive. Activity was apparently bound to the sulfonic substitution in the 3, 6 positions in the naphthalene rings, substitutions in the benzidine rings being not critical. All of the active and most of the inactive compounds were taken up by tubule cells of kidney cortex and by reticular and parenchymal cells of liver. While the effect on both liver and kidney was obtained 1 day after trypan blue administration, RNase remained increased for only about 3 days in the first organ, and for at least a month in the second. However, repeated trypan blue doses increased liver enzyme activity for at least 9 days. Serum RNase activity was decreased after trypan blue administration. Ethionine administration together with trypan blue markedly blocked the effect of the dye on liver RNase activity; simultaneously given methionine partially reversed the action of the antimetabolite. This suggests that de novo synthesis of RNase is induced in liver by trypan blue. The action of ethionine on the kidney RNase response to trypan blue was less marked although significant; in view of the possible kidney uptake of the plasma enzyme, interpretation of this finding must be postponed. Results are discussed with reference to the mechanism of the structural specificity of the compounds used, cytological localization of the dyes and their mechanism of action on liver and kidney RNase.     

Presence of a laminin-binding chondroitin sulfate proteoglycan at the cell surface of a human melanoma cell Mel-85

Working with Mel-85 (a human melanoma cell line), we have been able to detect a laminin-binding molecule with an apparent molecular mass of 100/110 kDa (Mel-85-LBM). Reduction with -mercaptoethanol decreases its molecular mass but does not affect its ability to bind laminin. This laminin interaction seems to be very specific since Mel-85-LBM binds laminin, but not fibronectin, vitronectin or type I collagen in affinity chromatography experiments. The molecule has a negative net charge at physiological pH and binds laminin in a divalent cation dependent way. Mel-85-LBM was metabolically radiolabeled with sodium [³⁵S]-sulfate and chemical -elimination of purified Mel-85-LBM releases chondroitin sulfate chains. Mel-85-LBM is also sensitive to chondroitinase ABC digestion. These findings show that this molecule is a chondroitin sulfate proteoglycan. The location of this proteoglycan at the cell surface is evidenced by experiments using a polyclonal antiserum raised against purified Mel-85LBM, that specifically reacts with just one molecule by western blotting among Mel-85 total cell extract as well as produces a positive signal by flow cytometry and a fluorescence profile of Mel-85 cells adhered on laminin.