Structure and Evolution of Genes Encoding Polyubiquitin and Ubiquitin-like Proteins in Arabidopsis Thaliana Ecotype Columbia

The Arabidopsis thaliana ecotype Columbia ubiquitin gene family consists of 14 members that can be divided into three types of ubiquitin genes; polyubiquitin genes, ubiquitin-like genes and ubiquitin extension genes. The isolation and characterization of eight ubiquitin sequences, consisting of four polyubiquitin genes and four ubiquitin-like genes, are described here, and their relationships to each other and to previously identified Arabidopsis ubiquitin genes were analyzed. The polyubiquitin genes, UBQ3, UBQ10, UBQ11 and UBQ14, contain tandem repeats of the 228-bp ubiquitin coding region. Together with a previously described polyubiquitin gene, UBQ4, they differ in synonymous substitutions, number of ubiquitin coding regions, number and nature of nonubiquitin C-terminal amino acid(s) and chromosomal location, dividing into two subtypes; the UBQ3/UBQ4 and UBQ10/UBQ11/UBQ14 subtypes. Ubiquitin-like genes, UBQ7, UBQ8, UBQ9 and UBQ12, also contain tandem repeats of the ubiquitin coding region, but at least one repeat per gene encodes a protein with amino acid substitutions. Nucleotide comparisons, K(s) value determinations and neighbor-joining analyses were employed to determine intra- and intergenic relationships. In general, the rate of synonymous substitution is too high to discern related repeats. Specific exceptions provide insight into gene relationships. The observed nucleotide relationships are consistent with previously described models involving gene duplications followed by both unequal crossing-over and gene conversion events.     

Opioid peptides in the nervous system ofAplysia: A combined biochemical,immunocytochemical, and electrophysiological study

Summary 1. We have used biochemical, immunocytochemical, and electrophysiological techniques to evaluate the role of opioid peptides in the central nervous system of the marine mollusc,Aplysia california.2. Binding studies using³H-d-Ala², met-enkephalinamide (³H-DAMA) showed a single class of high-affinity binding sites with aK _d of 1.3 nM and a binding density of 45 pmol/g.3. HPLC extracts of ganglia revealed multiple peaks with immunoreactivity for either leu (LEU-IR)- or met-enkephalin (MET-IR), but the amounts were not uniformly distributed in all ganglia.4. LEU-IR and MET-IR neurons were demonstrated immunocytochemically in all ganglia, but MET-IR neurons were more frequent and were concentrated in pedal and pleural ganglia. While absorption control studies abolished MET-IR, LEU-IR was only partially abolished in the neuropil.5. In electrophysiological studies, both depolarizing and hyperpolarizing responses were found tod-Ala²-leu-enkephalin (DALEU) andd-Ala²-met enkephalin (DAMET) on some and different neurons.6. HPLC fractions from regions with retention times corresponding to authentic leu- or met-enkephalin showed physiologic responses similar to those of DALEU and DAMET, respectively.7. These studies suggest that a variety of endogeneous opioid peptides play physiologically important roles in the nervous system ofAplysia, including but not necessarily limited to leu- and met-enkephalin.     

Interspecies recombination In nature: a meningococcus that has acquired a gonococcal PIB porin

A. vaginal isolate of Neisseria has been reported to resemble Neisseria meningitidis in biochemical characteristics but to react with serological reagents that are specific to the PI porin from Neisseria gonorrhoeae. We have confirmed that this isolate has the biochemical attributes of a meningococcus and have shown that it clusters among meningococcal Isolates on a dendrogram based on isoenzyme variation within housekeeping enzymes from populations of N. meningitidis and N. gonorrhoeae. Furthermore, the sequences of the fbp and adk genes were typical of those of N. meningitidis and were distinct from those of N. gonorrhoeae. However, the porB gene was very similar to the por genes of N. gonorrhoeae isolates that express the PIB class of outer-membrane porin (differing from one gonococcal por allele at only a single nucleotide site), and was clearly distinct from the porB genes of N. meningitidis. The isolate therefore appears to be a typical meningococcus, except that its porB gene has been replaced with the por gene from a gonococcus.     

Cell lineage patterns and homeotic gene activity during Antirrhinum flower development

Background: Homeotic genes controlling the identity of flower organs have been characterized in several plant species. To determine whether cells expressing these genes are specified to follow particular developmental fates, we have studied the pattern of cell lineages in developing flowers of Antirrhinum. Each flower has four whorls of organs, and progenitor cells of these can be marked at particular stages of development using a temperature-sensitive transposon. This allows the cell lineages in the flower to be followed, as well as giving information about rates of cell division.Results We show here that, prior to the emergence of organ primordia, cells in the floral meristem have not been allocated organ identities. After this time, lineage restrictions arise between whorls, correlating with the onset of expression of genes that control organ identity. A further lineage restriction appears slightly later on, between the dorsal and ventral surfaces of the petal. Our results further suggest that the rates of cell division fluctuate during key stages of meristem development, perhaps as a consequence of meristem-identity gene expression.Conclusion The patterns of lineage restriction and organ-identity gene expression in early floral meristems are consistent with some cells being allocated specific identities at about this stage of development. Plant cells cannot move relative to each other, so lineage restrictions in plants may reflect particular orientations and/or rates of growth at boundary regions.     

Kinetic properties of cholinergic desensitization in Aplysia neurons

The kinetic properties of desensitization onset of excitatory cholinergic responses were studied in isolated, voltage-clamped Aplysia neurons. Desensitization of the acetylcholine (ACh)-induced current in response to microperfused acetylcholine occurred in two phases, and was best modelled as the sum of two exponential components plus a constant. Both exponential components were accelerated by increasing ACh dose. At the higher ACh doses the current decline was dominated by the fast exponential component, and the ratio of the plateau-peak current was reduced. Over the range of membrane potentials -50 to -110 mV, no change in the kinetics of desensitization onset was observed. The mean time constants of both exponential components were doubled by cooling from 20 degrees C to 5 degrees C. These results demonstrate that, as at the vertebrate neuromuscular junction, the onset of desensitization of this ACh response involves at least two processes which are dose- and temperature-sensitive. The lack of voltage dependence contrasts with results from vertebrate preparations, and indicates a fundamental difference between the properties of the excitatory ACh response in Aplysia neurons and the vertebrate neuromuscular junction.     

Isolation of an evolutionarily conserved epidermal growth factor receptor cDNA from human A431 carcinoma cells

Complementary DNA corresponding to total poly(A)+-RNA from the human A431 epidermoid carcinoma cell line was cloned in the phage expression vector lambda gt 11. An epidermal growth factor (EGF) receptor cDNA clone was obtained by screening of the expression library with a rabbit polyclonal antibody (IgG), raised to the purified A431 EGF receptor, in combination with [125I]protein A of S. aureus. The cloned cDNA was able to select, by hybridization, messenger RNA which was translated in Xenopus oocytes and yielded an immunoprecipitable EGF receptor protein of Mr = 160,000. The insert of this cDNA (phEGFR-1), is approximately 880 base pairs in length and encodes the carboxyterminal portion of the EGF receptor protein. Its sequence is evolutionarily conserved among vertebrates as shown by hybridization to unique chromosomal DNA sequences from human, baboon, dog, rat, mouse and frog.     

Flow cytometric determination of radiation-induced chromosome damage and its correlation with cell survival

Chinese hamster M3-1 cells were irradiated with several doses of X rays or alpha particles from 238Pu. Propidium iodide-stained chromosome suspensions were prepared at different times after irradiation; cells were also assayed for survival. The DNA histograms of these chromosomes showed increased background counts with increased doses of radiation. This increase in background was cell-cycle dependent and was correlated with cell survival. The correlation between radiation-induced chromosome damage and cell survival was the same for X rays and alpha particles. Data are presented which indicate that flow cytometric analysis of chromosomes of irradiated cell populations can be a useful adjunct to classical cytogenic analysis of irradiation-induced chromosomal damage by virtue of its ability to express and measure chromosomal damage not seen by classical cytogenic methods.