The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants

The xylanase gene from Cryptococcus albidus contains seven introns. Genomic and cDNA clones under the control of the CaMV 35S promoter were transferred into tobacco plants using Agrobacterium-mediated cell transformation. The genes were transcribed and the mRNAs were amplified by the polymerase chain reaction using primers on each side of the intron region. About 90% of the amplification products from plants transformed with the genomic clone corresponded to the size of the pre-mRNA (1.2 kb) and 10% represented the spliced product (0.85 kb). The 0.85 kb fragment was cloned and sequenced and the result indicated that the introns from the xylanase gene were accurately spliced by the plant cells.     

Kinetics and subunit composition of NMDA receptors in respiratory-related neurons

NMDA receptors are involved in a variety of brainstem functions. The excitatory postsynaptic NMDA currents of pre-Botzinger complex interneurons and hypoglossal motoneurons, which are located in the medulla oblongata, show remarkably fast deactivation kinetics of approximately 30 ms compared with NMDA receptors in other types of neurons. Because structural heterogeneity might be the basis for physiological properties, we examined the expression of six NMDA receptor subunits (NMDAR1, NR2A-2D, and NR3A) plus eight NMDR1 splice variants in pre-Botzinger complex, hypoglossal and, for comparison, neurons from the nucleus of the solitary tract in young rats using single cell multiplex RT-PCR. Expression of NR2A, NR2B, and NR2D was observed in all three cell types while NR3A was much more abundant in pre-Botzinger complex interneurons, which belong to the rhythm generator of respiratory activity. In hypoglossal neurons, the NMDAR1 splice variants NMDAR1-4a and NMDAR1-4b were found. In neurons of the nucleus of the solitary tract, instead of NMDAR1-4b, the NMDAR1-2a splice variant was detected. This differential expression of modulatory splice variants might be the molecular basis for the characteristic functional properties of NMDA receptors, as neurons expressing a special NMDAR1 splice variant at the mRNA level show fast kinetics compared with neurons lacking this splice variant.     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

Effect of cytokinins on ribosomal RNA gene expression in excised cotyledons of Cucurbita pepo L.

Excised pumpkin (Cucurbita pepo L.) cotyledons were used to investigate the effects of two different types of cytokinins: N⁶-benzyladenine and N₁-(2-chloro-4-pyridyl)-N₂-phenylurea on RNA synthesis in isolated nuclei. Treatment of cotyledons with both cytokinins resulted in a rapid enhancement of nuclear RNA-polymerase-I activity (EC 2.7.7.6). Maximum stimulation of RNA polymerase I, responsible for rRNA synthesis, was observed 4–6 h after the start of cytokinin action. The activity of RNA polymerase II was stimulated much more slowly and to a lesser extent. Uridine 5-monophosphate-uridine analysis of the alkalidigested nascent pre-rRNA chains showed that the stimulation of RNA-polymerase-I activity was the consequence of an increase of the polyribonucleotide-clongation rate. No significant change in the number of transcribing enzyme molecules was defected after hormone treatment (86·10³ RNA-polymerase-I molecules per diploid genome).Indications that de-novo protein synthesis is necessary for cytokinin-mediated RNA-polymerase stimulation were derived from experiments showing inhibition by cycloheximide.Abbreviations BA N⁶-benzyladenine - [PU]-30 N₁-(2-chloro-4-pyridyl)-N₂-phenylurea - UMP undine 5-monophosphate - UTP udine 5-triphosphate     

A direct approach to the measurement of genetic variation in fish populations: applications of the polymerase chain reaction to studies of Atlantic cod, Gadus morhua L.

We used the polymerase chain reaction (PCR) and direct DNA sequencing to study genetic variation within and among populations of Atlantic cod, Gadus morhua , in the western North Atlantic. In a 307 bp region of the mitochondrial cytochrome b gene, 24 variable nucleotide positions define 24 genotypes, which differ by from one to six nucleotide substitutions. Greenland cod ( G. ogac ) differs from the most similar G. morhua genotype by an additional 12 nucleotide substitutions. Silent transitions dominate both intra- and interspecific comparisons, however four nucleotide substitutions within morhua result in amino acid replacements. Direct sequencing of DNA reveals substantially more of the genetic variation that exists within and between species than do previous indirect methods based on restriction fragment length polymorphisms, and thus has far greater potential to quantify such differences as may exist among fish stocks. Preliminary experiments also indicate that automation of DNA sequencing provides an efficient, rapid, and accurate means for detection of genetic variation in natural populations offish.     

Mitochondrial malate dehydrogenase from watermelon: sequence of cDNA clones and primary structure of the higher-plant precursor protein

The isolation and sequence of a cDNA clone encoding the complete mitochondrial malate dehydrogenase (mMDH) of watermelon cotyledons is presented. Taking advantage of the polymerase chain reaction technology partial cDNA clones from the central part, the 3 part and the 5 part of the mRNA were obtained with oligonucleotides based on directly determined amino acid sequences. Subsequently, two complete cDNA clones for mMDH were synthesized with a sense primer corresponding to the nucleotide sequence of the amino terminal end of pre-mMDH and two antisense primers corresponding to the major alternative adenylation sites found in the mRNA.The amino acid residues for substrate and cofactor binding identified by X-ray crystallography for pig heart cytoplasmic MDH are conserved in the 320 amino acid long mature higher-plant mMDH. A presequence of 27 amino acids is present at the amino terminal end of the precursor protein.     

Frequency of infection with A and B supergroup <Emphasis Type="Italic">Wolbachia</Emphasis> in insects and pests associated with mulberry and silkworm

Wolbachia is a ubiquitous, Gram-negative, vertically transmitted, alpha-proteobacterium that causes an array of reproductive abnormalities including cytoplasmic incompatibility, feminization of genetic males, parthenogenesis in a number of insect species, among others. Wolbachia is now being exploited as an agent for pest and vector control. Previous surveys indicated that it is commonly seen in 16–76% of arthropods. In this paper, using polymerase chain reaction assay based on specific amplification of the ftsZ-A and-B supergroup Wolbachia gene fragments, we found that 30% of insects and pests screened were positive for Wolbachia. Among them 66.7% harbour double Wolbachia infection, while 33.3% harbour single Wolbachia infection. These results indicate widespread infection with both double and single Wolbachia, and provide a wealth of information to exploit this endobacterium for the management of pests and vectors.