Successful infection of the common marmoset (Callithrix jacchus) with human varicella-zoster virus.

The common marmoset, Callithrix jacchus, can be infected with human varicella-zoster virus (VZV), both wild-type strain KMcC and attenuated vaccine strain Oka/Merck. Infection was accomplished with either whole-cell-associated or cell extract VZV by combined oral-nasal-conjunctival application and was characterized by substantial and persistent anti-VZV antibody responses. The infectivity of VZV for marmosets was destroyed by treatment of inocula with heat or UV light. Diluted inocula with as few as 40 PFU/ml were infectious for marmosets. The lungs were demonstrated to be a major site of viral replication; both the presence of viral antigens and signs of pneumonia were demonstrated in lung tissues. Four serial passages of VZV KMcC were carried out in C. jacchus by a process of in vitro isolation and culturing of VZV from infected lung tissue and reapplication of the cultured isolates to fresh animals. The isolated viruses were identified as VZV both serologically and by restriction endonuclease analyses. The C. jacchus infectivity model should prove useful for determining the efficacy of subunit and live recombinant VZV vaccines as well as for the study of zoster.     

A nomenclature for the genes encoding the chlorophylla/b-binding proteins of higher plants

We propose a nomenclature for the genes encoding the chlorophylla/b-binding proteins of the light-harvesting complexes of photosystem I and II. The genes encoding LHC I and LHC II polypeptides are namedLhca1 throughLhca4 andLhcb1 throughLhcb6, respectively. The proposal follows the general format recommended by the Commision on Plant Gene Nomenclature. We also present a table for the conversion of old gene names to the new nomenclature.     

HeLa cell mutants resistant to epidermal growth factor ricin A-chain conjugate

Epidermal growth factor (EGF) was linked to the toxic A chain of ricin toxin (RTA) to produce an EGF-receptor-specific cytotoxic agent, EGF-RTA. Three EGF-RTA-resistant mutants of the human HeLa cell line were selected. These mutant cell lines are 10-fold to more than 100-fold more resistant to EGF-RTA when compared to HeLa cells. The EGF-RTA-resistant mutants have at least as many EGF receptors as parent cells; the basis for the EGF-RTA-resistant phenotype must be distal to EGF binding. The EGF-RTA-resistant cells are not cross-ressitant to ricin or to diphtheria toxin; their mutant phenotype appears to be EGF specific. The EGF-RTA-resistant mutants are able to internalize and degrade EGF. However, the mutants have altered EGF receptor down-regulation and phorbol 12-tetradecanoate 13-acetate modulation properties. EGF-RTA/ammonium chloride and EGF-RTA/adenovirus co-treatment data suggest that the mutant defect(s) which confers EGF-RTA resistance is either in the endosome or at a step(s) in the intracellular EGF processing pathway between the endosome and the lysosome.     

Identifying DNA polymorphisms in human TCRA/D variable genes by direct sequencing of PCR products

 The T-cell receptor (TCR) is a highly variable molecule composed of two polypeptide chains that recognize antigenic peptides in the context of major histocompatibility complex (MHC) molecules. In this study, we describe a sequence-based search for germline polymorphisms in the variable (V) gene segments of the human TCRA/D locus. Thirty different V gene segments were amplified from six to eight unrelated individuals and sequenced from low melting point agarose. Twenty-seven polymorphisms were identified in 15 V gene segments. These polymorphisms are mainly single nucleotide substitutions, but an insertion/deletion polymorphism and a single dinucleotide repeat with variable length were also seen. Of the 15 sequence variations found in the coding regions, six are silent and nine encode amino acid changes. All of the amino acid changes are found at non-conserved residues, frequently in the hypervariable regions, where they may influence MHC and/or peptide recognition. Therefore, it is possible that germline variations in TCR genes could influence an individual’s immune response, and may also contribute to susceptibility to diseases such as autoimmunity. Received: 9 January 1996 / Revised: 22 February 1996     

Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein

The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O₂ evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217–227).     

Restriction fragment length polymorphisms distinguish among accessions ofCeratopteris thalictroides andC. richardii (Parkeriaceae)

We have used cDNA clones as probes on Southern blots to detect restriction fragment length polymorphisms among sevenCeratopteris thalictroides accessions, threeC. richardii accessions, and one putative interspecific hybrid. We found that the stringency of post-hybridization washes was a critical parameter affecting the quality of our blots; even with homologous cDNA sequences low stringency conditions resulted in a smear of signal, but high stringency washes gave blots with distinct bands. Most probes showed hybridization with four or more genomic fragments. Similarities in the number and size of fragments between and within species indicated that (i)C. richardii shows limited polymorphism among accessions tested, (ii)C. thalictroides is highly polymorphic, and (iii) Hawaiian accessions ofC. thalictroides are divergent relative to their continental cohorts and among themselves. The putative interspecific hybrid did not group closely with either of these species.