Hypervariable region IV of Salmonella gene fliCd encodes a dominant surface epitope and a stabilizing factor for functional flagella.

To identify the major antigenic determinant of native Salmonella flagella of antigenic type d, we constructed a series of mutated fliCd genes with deletions and amino acid alterations in hypervariable region IV and in region of putative epitopes as suggested by epitope mapping with synthetic octameric peptides (T.M. Joys and F. Schödel, Infect. Immun. 59:3330-3332, 1991). The expressed product of most of the mutant genes, with deletions of up to 92 amino acids in region IV, assembled into functional flagella and conferred motility on flagellin-deficient hosts. Serological analysis of these flagella with different anti-d antibodies revealed that the peptide sequence centered at amino acids 229 to 230 of flagellin was a dominant B-cell epitope at the surface of d flagella, because replacement of these two amino acids alone or together with their flanking sequence by a tripeptide specified by a linker sequence eliminated most reactivity with antisera against wild-type d flagella as tested by enzyme-linked immunosorbent assay or by Western immunoblot. Functional analysis of the mutated flagellin genes with or without an insert suggested that amino acids 180 to 214 in the 5' part of hypervariable region IV (residues 181 to 307 of the total of 505) is important to the function of flagella. The hybrid proteins formed by insertion of peptide sequence pre-S1 12-47 of hepatitis B virus surface antigen into the deleted flagellins assembled into functional flagella, and antibody to the pre-S1 sequence was detected after immunization of mice with the hybrid protein. This suggests that such mutant flagellins containing heterologous epitopes have potential as vaccines.     

Compositional heterogeneity of the Escherichia coli genome: A role for VSP repair?

E. coli genes that contain a high frequency of the tetranucleotide CTAG are also rich in the tetramers CTTG, CCTA, CCAA, TTGG, TAGG, and CAAG (group-I tetramers). Conversely, E. coli genes lacking CTAG are rich in the tetranucleotides CCTG, CCAG, CTGG, and CAGG (group-II tetramers). These two gene samples differ also in codon usage, amino acid composition, frequency of Dcm sites, and contrast vocabularies. Group-I tetramers have in common that they are depleted by very-short-patch repair (VSP), while group-II tetramers are favored by VSP activity. The VSP system repairs G:T mismatches to G:C, thereby increasing the overall G+C content of the genome; for this reason the CTAG-rich sample has a lower G+C content than the CTAG-poor sample. This compositional heterogeneity can be tentatively explained by a low level of VSP activity on the CTAG-rich sample. A negative correlation is found between the frequency of group-I tetramers and the level of gene expression, as measured by the Codon Adaptation Index (CAI). A possible link between the rate of VSP activity and the level of gene expression is considered.Correspondence to: A. Marine     

Intracerebroventricular Administration of AT1 Receptor Antisense Oligonucleotides Inhibits the Behavioral Actions of Angiotensin II

Abstract: Antisense Oligonucleotides were developed to study the expression and function of angiotensin type 1 (AT₁) receptors in cultured cells and brain. In both liver epithelial WB and neuro-blastoma N1E-115 cells AT₁ antisense oligomers substantially decreased AT₁ receptor density, whereas angiotensin type 2 (AT₂) receptors remained unchanged. Similarly, repeated intracerebroventricular injections of AT₁ antisense oligomers in rats decreased AT₁ receptor density in hypothalamic-thalamic-septal tissue, and AT₂ receptors were unaffected. Intracerebroventricular antisense oligomers also attenuated drinking elicited by intra-cerebroventricular angiotensin II but not the cholinomimetic carbachol. Collectively, these results demonstrate that antisense Oligonucleotides attenuate angiotensin receptor expression and function in behaving animals.     

Chloroplast DNA inheritance in theStellaria longipes complex (Caryophyllaceae)

Inheritance of chloroplast DNA (cpDNA) was examined in F₁ progenies derived from three crosses and three corresponding reciprocal crosses betweenStellaria porsildii andS. longifolia. Chloroplast DNA restriction fragments were analyzed using methods of nonradioactive digoxigenin-11-dUTP labeling and chemiluminescent detection with Lumi-Phos 530. Distinct interspecific restriction fragment polymorphisms were identified and used to demonstrate the mode of cpDNA inheritance. Mode of cpDNA inheritance differed among crosses. Two crosses in whichS. porsildii, SP2920-21, was the maternal parent exhibited three different types of plastids, maternal, paternal and biparental, among the F₁ hybrids, suggesting a biparental cpDNA inheritance and plastid sorting-out inStellaria.     

RFLP tagging of a new semidwarfing gene in rice

A new rice semidwarfing gene which is not allelic tosd1, temporarily designated assdg, might be of use as a new source of semidwarfism in rice breeding programs. We report here the identification of a DNA marker closely linked to this gene. The DNA marker was identified by testing 120 mapped rice RFLP makers as hybridization probes for Southern analysis of a pair of nearly isogenic lines with or withoutsdg. Linkage association of the marker with the gene was verified using a F₂ population segregating for semidwarfism. RFLP analysis showed thatsdg is closely linked to a single-copy DNA clone RZ182 on chromosome 5, with a distance of 4.3 centiMorgans between them. This marker may facilitate early selection for the semidwarfing gene in rice breeding programs     

Spontaneous hybridization between a male-sterile oilseed rape and two weeds

Spontaneous interspecific hybrids were produced under natural conditions (pollination by wind and bees) between a male-sterile cybrid Brassica napus (AACC, 2n = 38) and two weeds Brassica adpressa (AdAd, 2n = 14) and Raphanus raphanistrum (RrRr, 2n = 18). After characterization by chromosome counts and isozyme analyses, we observed 512 and 3 734 inter-specific seeds per m² for the B. napus-B. adpressa and B. napus-R. raphanistrum trials respectively. Most of the hybrids studied had the expected triploid structure (ACX). In order to quantify the frequency of allosyndesis between the genomes involved in the hybrids, their meiotic behavior was compared to a haploid of B. napus (AC). For the B. napus-B. adpressa hybrids, we concluded that probably no allosyndesis occurred between the two parental genomes, and that genetic factors regulating homoeologous chromosome pairing were carried by the B. adpressa genome. For the B. napus-R. raphanistrum hybrids, high chromosome pairing and the presence of multivalents (in 9.16% of the pollen mother cells) indicate that recombination is possible between chromosomes of different genomes. Pollen fertility of the hybrids ranged from 0 to 30%. Blackleg inoculation tests were performed on the three parental species and on the interspecific hybrids. BC₁ production with the weeds and with rapeseed was attempted. Results are discussed in regard to the risk assessment of transgenic rapeseed cultivation, F₁ hybrid rapeseed variety production, and rapeseed improvement.     

Carbon dioxide consumption during soil development

Carbon is sequestered in soils by accumulation of recalcitrant organic matter and by bicarbonate weathering of silicate minerals. Carbon fixation by ecosystems helps drive weathering processes in soils and that in turn diverts carbon from annual photosynthesis-soil respiration cycling into the long-term geological carbon cycle. To quantify rates of carbon transfer during soil development in moist temperate grassland and desert scrubland ecosystems, we measured organic and inorganic residues derived from the interaction of soil biota and silicate mineral weathering for twenty-two soil profiles in arkosic sediments of differing ages. In moist temperate grasslands, net annual removal of carbon from the atmosphere by organic carbon accumulation and silicate weathering ranges from about 8.5 g m^–2 yr^–1 for young soils to 0.7 g M^–2 yr^–1 for old soils. In desert scrublands, net annual carbon removal is about 0.2 g m^–2 yr^–1 for young soils and 0.01 g m^–2 yr^–1 for old soils. In soils of both ecosystems, organic carbon accumulation exceeds CO₂ removal by weathering, however, as soils age, rates of CO₂ consumption by weathering accounts for greater amounts of carbon sequestration, increasing from 2% to 8% in the grassland soils and from 2% to 40% in the scrubland soils. In soils of desert scrublands, carbonate accumulation far outstrips organic carbon accumulation, but about 90% of this mass is derived from aerosolic sources that do not contribute to long-term sequestration of atmospheric carbon dioxide.     

Characterization of the polyadenylation signal of influenza virus RNA.

It has been shown that a stretch of uridines (U's) near the 5' end of the virion RNA of influenza A virus is the polyadenylation site for viral mRNA synthesis. In addition, the RNA duplex made up the 3' and 5' terminal sequences adjacent to the U stretch is also involved in polyadenylation. We have further characterized the polyadenylation signal of influenza virus RNA with a ribonucleoprotein transfection system. We found that the optimal length of the U stretch is 5 to 7 uridine residues. We also showed that the upstream sequence at the 5' end is not involved in polyadenylation and that the optimal distance between the 5' end and the U stretch is 16 nucleotides. The combination of these features defines the polyadenylation site and differentiates this signal from other U stretches scattered throughout the genomes of influenza viruses.     

Capsid assembly and involved function analysis of twelve core protein mutants of duck hepatitis B virus.

The roles of different regions of the duck hepatitis B virus (DHBV) core protein on viral capsid assembly and related functions were examined. Twelve deletion and insertion mutations which covered 80% of the DHBV C open reading frame were constructed and expressed in Escherichia coli. The N-terminal region (amino acids 3 to 66) of DHBV core protein was important for its tertiary structure and function in E. coli. The expressed core mutants without this region apparently inhibited E. coli growth. The results of transmission electron microscopy of E. coli thin sections, capsid agarose gel, and sucrose gradient sedimentation demonstrated that a few DHBV core mutants with insertion in the N terminus and deletion in the C terminus retained the ability to form core-like particles in E. coli. However, other mutations in most of N-terminal and central regions strongly inhibited the self-assembly ability of DHBV core protein in E. coli. In addition, the mutant with a C-terminal region deletion (amino acids 181 to 228) lost most of the nucleic acid-binding activity of the DHBV core protein.     

Requirement of the Pr55gag precursor for incorporation of the Vpr product into human immunodeficiency virus type 1 viral particles.

The human immunodeficiency virus type 1 (HIV-1) particles consists of two molecules of genomic RNA as well as molecules originating from gag, pol, and env products, all synthesized as precursor proteins. The 96-amino-acid Vpr protein, the only virion-associated HIV-1 regulatory protein, is not part of the virus polyprotein precursors, and its incorporation into virus particles must occur by way of an interaction with a component normally found in virions. To investigate the mechanism of incorporation of Vpr into the HIV-1 virion, Vpr- proviral DNA constructs harboring mutations or deletions in specific virion-associated gene products were cotransfected with Vpr expressor plasmids in COS cells. Virus released from the transfected cells was tested for the presence of Vpr by immunoprecipitation with Vpr-specific antibodies. The results of these experiments show that Vpr is trans-incorporated into virions but at a lower efficiency than when Vpr is expressed from a proviral construct. The minimal viral genetic information necessary for Vpr incorporation was a deleted provirus encoding only the pr55gag polyprotein precursor. Incorporation of Vpr requires the expression but not the processing of gag products and is independent of pol and env expression. Direct interaction of Vpr with the Pr55gag precursor protein was demonstrated by coprecipitation experiments with gag product-specific antibodies. Overall, these results indicate that HIV-1 Vpr is incorporated into the nascent virion through an interaction with the Gag precursor polyprotein and demonstrate a novel mechanism by which viral protein can be incorporated into virus particles.