Studies on the maintenance and expression of cloned DNA fragments in the nuclear genome of the green alga Chlamydomonas Reinhardtii

Using a biolistic device built here and based on the principle of the device described by Klein et al. (1987). we have reproducibly obtained transformants of Chlamydomonas reinhardtii . The reproducibility of the method has allowed us to examine the maintenance and expression of cloned DNA fragments introduced into C. Reinhardtii .     

Locus Heterogeneity for Waardenburg Syndrome is Predictive of Clinical Subtypes

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between −2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.     

Analysis of γ-Aminobutyric AcidA Receptor Subunits in the Mouse Cochlea by Means of the Polymerase Chain Reaction

Abstract: Unlike 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces consistent decreases in levels of striatal dopamine (DA) with considerably smaller and more variable effects on mouse brain levels of serotonin (5-HT) and norepinephrine (NE), a novel amine-substituted MPTP analogue, 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH₂-MPTP), administered in a standard mouse dosing paradigm for MPTP (20 mg/kg X 4) did not affect striatal DA but led to marked reductions (60–70%) in levels of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and NE measured in frontal cortex and hippocampus 1 week after treatment. Another 2'-substituted MPTP analogue, 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine, affected cortical and hippocampal 5-HT, 5-HIAA, and NE only minimally, while markedly reducing the DA content in striatum (90%), thus indicating that the substituent (-NH₂ versus -CH₃) at the 2'position is important for the differential effects of these MPTP analogues. In a replication study with a 3-week end point, hippocampal and cortical 5-HT, 5-HIAA, and NE levels remained depressed with no indication of recovery. These results suggest that 2'-NH₂-MPTP may be a novel, regionally selective neurotoxin for serotonergic and norad-renergic nerve terminals.     

Large arrays of tandemly repeated DNA sequences in the green alga Chlamydomonas reinhardtii

We describe the characterization of tandemly repeated DNA sequences, which resemble the satellite DNA sequences of multicellular eucaryotes, in the unicellular green alga Chlamydomonas reinhardtii. Restriction enzymes that cleave C. reinhardtii DNA relatively frequently produce a number of high molecular weight DNA fragments in addition to the bulk of low molecular weight DNA fragments. pTANC 1.5 contains a 1.5 kb Sau3A fragment cloned from one of these large bands. pTANC 1.5 hybridized to at least three large arrays (200 to 700 kb) of tandemly repeated DNA sequences in the cell-wall-deficient strain cw1.5. These arrays are composed of repeat units that are each cleaved once by BamHl into bands of 1.5, 1.9, 2.0 and 2.5 kb in size. The copy numbers of the 1.5, 1.9, 2.0 and 2.5 kb Bamhl bands vary between different C. reinhardtii strains. Chlamydomonas smithii and a number of C. reinhardtii strains are deficient in all four BamHl bands. Genetic analysis of wild-type strain 137c, which is deficient in the 2.0 kb BamHl band, indicates that the 1.5, 1.9 and 2.5 kb BamHl bands derive from at least five loci. The 1.5, 1.9 and 2.5 kb repeat units are not extensively interspersed with each other in strain 137c. Pulsed-field gel electrophoresis of intact C. reinhardtii chromosomes indicates that TANC arrays are present on more than one chromosome.     

Structural insights into thermostable direct hemolysin of Vibrio parahaemolyticus using single-particle cryo-EM

Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)—the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2 Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3 Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.     

Germinating pigeonpea (Cajanus cajan) seeds secrete factor(s) having antiethylene-like effects

Germinating pigeonpea seeds were found to release factor(s), which dramatically altered the growth pattern of rice seedlings. This included an increased sensitivity of roots to gravity, an increase in seminal root length, and increased density of branch roots on the seminal root and suppression of root hair formation. Furthermore, the known characteristic responses of rice to ethylene (namely, coleoptile growth promotion and induction of senescence) were inhibited by the presence of these factor(s). This altered growth pattern of rice seedlings was similar to that brought about by the ethylene-antagonist, silver nitrate. Further, the change in growth pattern in rice was specifically reversed by ethrel. In addition, in wild-type Arabidopsis thaliana these factors suppressed formation of root hair, the development of which is known to be promoted by ethylene. In contrast, in a mutant A. thaliana which shows constitutive ethylene response ( ctr1-1 ), the factors failed to inhibit root hair formation. These observations indicated that germinating pigeonpea seeds secrete factor(s), which have antiethylene-like effects.     

Inhibition of Kinesin Synthesis In Vivo Inhibits the Rapid Transport of Representative Proteins for Three Transport Vesicle Classes into the Axon

Abstract: We have previously demonstrated that the in vivo vitreal injection of an antisense oligonucleotide directed to the kinesin heavy chain inhibits retinal kinesin synthesis by 82% and concomitantly inhibits rapid transport of total protein into the optic nerve by 70%. These results establish a major role for kinesin in rapid axonal transport in vivo. Recently, the cloning of a family of kinesin-like molecules from the mammalian brain has been reported, and some of these proteins are also expressed in neurons. To assign a specific function to the kinesin heavy chain we inhibited the kinesin synthesis with an antisense kinesin oligonucleotide and assessed the axonal transport into the optic nerve of representative proteins from each of three vesicle classes that contain rapidly transported proteins. Marker proteins used were substance P for peptide-containing synaptic vesicles, the amyloid precursor protein for plasma membrane precursor vesicles, and several integral synaptic vesicle proteins. Our results indicate that the major anterograde motor protein for all three vesicle classes utilizes kinesin heavy chain, although we discuss alternative explanations.     

Immunological evidence for accumulation of two high-molecular-weight (104 and 90 kDa) HSPs in response to different stresses in rice and in response to high temperature stress in diverse plant genera

Rice seedlings accumulate stainable amounts of the 104 and 90 kDa polypeptides in response to high temperature stress. We have purified and raised highly specific polyclonal antisera against both of these polypeptides. In western blotting experiments, we find that these proteins are accumulated to different extents in rice seedlings subjected to salinity (NaCl), water stress, low-temperature stress and exogenous abscisic acid application. These proteins also accumulated when rice seedlings grown in pots under natural conditions were subjected to water stress by withholding watering. Seedlings of Triticum aestivum, Sorghum bicolor, Pisum sativum, Zea mays, Brassica juncea and mycelium of Neurospora crassa showed accumulation of the immunological homologues of both the 104 and the 90 kDa polypeptides, in response to high-temperature stress. We have earlier shown that shoots of rice seedlings exposed to heat shock accumulate a 110 kDa polypeptide which is an immunological homologue of the yeast HSP 104 (Singla and Grover, Plant Mol Biol 22: 1177–1180, 1993). Employing anti-rice HSP 104 antibodies and anti-yeast HSP 104 antibodies together, we provide evidence that rice HSP 104 is different from the earlier characterized rice HSP 110.     

Occurrence of Diaphanosoma excisum (Sars) on a sandy beach at Balramgari (Orissa), India

The occurrence of Diaphanosoma excisum (Sars), a freshwater cladoceran, in benthic samples of an intertidal sandy beach is reported. Population density was seasonal. A relatively high density was recorded from June to September (south-west monsoon season) with a maximum (46 ind 10 cm^–2) in September at a depth of 10–15 cm of sediment. A sudden decline occurred during north-east monsoon (October to January), and in the fair season (February to May), the cladocerans disappeared. Mean density varied (P<0.001) with sediment depth and season and showed a contagious dispersion. Abundance was negatively correlated with salinity (r = –0.76) but positively with POC (r =0.79) and mean grain size of the sediment (r = 0.93). The density of D. excisum was highest in fine sand.