Cloning of the Promoter Regions of Mouse TGF-{beta} Receptor Genes by Inverse PCR with Highly Overlapped Primers

In order to isolate promoters of mouse TGF-ß receptorgenes, we used inverse PCR with highly overlapped primers correspondingto the 5' sequence of the receptor cDNAs. Nested primer setsonly covered a 30- to 40-base region of the sequences. HinfI-digestedand self-ligated mouse genomic DNA was used as a PCR template.Only one band for each receptor was seen after PCR. The amplifiedDNA fragments could direct luciferase production when the luciferasecoding sequence was ligated after the fragments. The sequenceof the fragment which correspond to the type II receptor showedpartial homology with the promoter region of the human TGF-ßtype II receptor. Thus, the inverse PCR with highly overlappedprimers could be an easy way to isolate the promoter regionsof many genes.     

Effects of soil water conditions on the morphology, phenology, and photosynthesis of Betula ermanii in the boreal forest

Four 2-year watering treatments were applied to Betula ermanii seedlings to investigate their responses to soil water conditions: ID [irrigated well (I) in the previous year and water deficit (D) in the current year], DD, DI, and II. RGR of the seedlings in current-year water deficit without experiencing previous-year water deficit (ID) was smaller than that of seedlings irrigated well in both years (II). Surprisingly, RGR did not differ between 2-year-water-deficited (DD) and -well-irrigated (II) treatments. There was no difference in the area-based photosynthetic rate of the late leaves, which are formed in the current-year environment, between the four water treatments, but their leaf longevity was shortened by water deficit. Area-based photosynthetic rate of the early leaves, whose buds are formed in the previous year environment, was smaller in ID than in II, but it did not differ between DD and II. The de-epoxidation state of the xanthophyll cycle was greater in ID than in DD. Leaf mass per area (leaf dry weight/leaf area) did not differ among the four water treatments. Specific root length (root total length/root dry weight) did not differ between ID and II. It was greater in DD than in II, probably resulting in increased water uptake efficiency. Photoprotective systems such as xanthophyll contents and antioxidant enzyme activities did not differ among the four treatments. Morphological responses of B. ermanii roots seem to be important as whole-plant-level responses to water deficit for maintaining RGR, in addition to leaf-level photosynthetic and phenological responses of two types of leaves. Our results partly explain how B. ermanii seedlings survive and maintain growth even under varying soil water conditions in the boreal forest.     

Localization of the gene encoding the human L-glutamate transporter (GLT-1) to 11p11.2–p13 by fluorescence in situ hybridization

High affinity glutamate transporters regulate levels of extracellular glutamate in the central nervous system. Impaired glutamate transport has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). The glutamate transporter subtypes GluT-1 and EAAC1 have previously been mapped to human chromosomes 5p13 and 9p24, respectively. In the present study, the GLT-1 subtype was mapped to human chromosome 11p11.2–p13 by fluorescence in situ hybridization. The possible clinical implications of this finding are discussed.     

A role for RAD54B in homologous recombination in human cells.

In human somatic cells, homologous recombination is a rare event. To facilitate the targeted modification of the genome for research and gene therapy applications, efforts should be directed toward understanding the molecular mechanisms of homologous recombination in human cells. Although human genes homologous to members of the RAD52 epistasis group in yeast have been identified, no genes have been demonstrated to play a role in homologous recombination in human cells. Here, we report that RAD54B plays a critical role in targeted integration in human cells. Inactivation of RAD54B in a colon cancer cell line resulted in severe reduction of targeted integration frequency. Sensitivity to DNA-damaging agents and sister-chromatid exchange were not affected in RAD54B-deficient cells. Parts of these phenotypes were similar to those of Saccharomyces cerevisiae tid1/rdh54 mutants, suggesting that RAD54B may be a human homolog of TID1/RDH54. In yeast, TID1/RDH54 acts in the recombinational repair pathway via roles partially overlapping those of RAD54. Our findings provide the first genetic evidence that the mitotic recombination pathway is functionally conserved from yeast to humans.     

Inhibitory effects of dorsal horn and excitant effects of ventral horn intraspinal microinjections of norepinephrine and serotonin in the cat

Norepinephrine and serotonin microinjections (10 μg) into the dorsal spinal gray matter depressed C-fiber reflexes induced by electrical stimulation of the superficial peroneal nerve or radiant heat stimulation of the footpad. Microinjection of norepinephrine or serotonin into the ventral gray matter facilitated C-fiber reflexes. These studies lend support to the suggestion that dorsal horn projections of neuronal systems which utilized norepinephrine or serotonin as neurotransmitters inhibit nociceptive spinal reflexes, and ventral horn projections facilitate spinal reflexes.