Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.     

The primary structure of the alpha subunit of human elongation factor 1. Structural aspects of guanine-nucleotide-binding sites

The primary structure of the alpha subunit of elongation factor 1 (EF-1 alpha) from human MOLT 4 cells was determined by cDNA sequencing. The data show that the conservation of the amino acid sequence is more than 80% when compared with yeast and Artemia EF-1 alpha. An inventory of amino acid sequences around the guanine-nucleotide-binding site in elongation factor Tu from Escherichia coli and homologous amino acid sequences in G proteins, initiation and elongation factors and proteins from the RAS family shows two regions containing conserved sequence elements. Region I has the sequence apolar-Xaa-Xaa-Xaa-Gly-Xaa-Xaa-Yaa-Xaa-Gly-LYs-Thr(Ser)- -Xaa-Xaa-Xaa-Xaa-X-apolar. Except for RAS proteins, Yaa is always an acidic amino acid residue. Region II is characterized by the invariant sequence apolar-apolar-Xaa-Xaa-Asn-Lys-Xaa-Asp. In order to facilitate sequence comparison we have used a graphic display, which is based on the hydrophilicity values of individual amino acids in a sequence.     

Wet cleaving of cells: A method to introduce macromolecules into the cytoplasm : Application for immunolocalization of cytosol-exposed antigens

A new method, called "wet cleaving," has been introduced to allow direct exposure of cytoplasm to externally supplied macromolecules by mechanical rupture of the plasma membrane. Monolayers of adherent cells or poly-L-lysine-attached suspension cells are overlayed with nitrocellulose sheets. By subsequent removal of the sheets, cells are cleaved, thereby exposing the cytoplasm. The method allows bulk quantities of cells to be cleaved in an efficient manner. Cleavage, although imposing some mechanical stress on the cells, leaves most if not all organelles morphologically intact, as shown by electron microscopy. Mechanically ruptured cells are well suited for use in immunocytochemical studies, as is demonstrated with the immunofluorescence localization of vinculin in chicken embryo fibroblasts.     

Evolutionary transfer of ORF-containing group I introns between different subcellular compartments (chloroplast and mitochondrion)

We describe here a case of homologous introns containing homologous open reading frames (ORFs) that are inserted at the same site in the large subunit (LSU) rRNA gene of different organelles in distantly related organisms. We show that the chloroplast LSU rRNA gene of the green alga Chlamydomonas pallidostigmatica contains a group I intron (CpLSU.2) encoding a site-specific endonuclease (I-CpaI). This intron is inserted at the identical site (corresponding to position 1931-1932 of the Escherichia coli 23S rRNA sequence) as a group I intron (AcLSU.m1) in the mitochondrial LSU rRNA gene of the amoeboid protozoon Acanthamoeba castellanii. The CpLSU.2 intron displays a remarkable degree of nucleotide similarity in both primary sequence and secondary structure to the AcLSU.m1 intron; moreover, the Acanthamoeba intron contains an ORF in the same location within its secondary structure as the CpLSU.2 ORF and shares with it a strikingly high level of amino acid similarity (65%; 42% identity). A comprehensive survey of intron distribution at site 1931 of the chloroplast LSU rRNA gene reveals a rather restricted occurrence within the polyphyletic genus Chlamydomonas, with no evidence of this intron among a number of non- Chlamydomonad green algae surveyed, nor in land plants. A parallel survey of homologues of a previously described and similar intron/ORF pair (C. reinhardtii chloroplast CrLSU/A. castellanii mitochondrial AcLSU.m3) also shows a restricted occurrence of this intron (site 2593) among chloroplasts, although the intron distribution is somewhat broader than that observed at site 1931, with site-2593 introns appearing in several green algal branches outside of the Chlamydomonas lineage. The available data, while not definitive, are most consistent with a relatively recent horizontal transfer of both site-1931 and site- 2593 introns (and their contained ORFs) between the chloroplast of a Chlamydomonas-type organism and the mitochondrion of an Acanthamoeba- like organism, probably in the direction chloroplast to mitochondrion. The data also suggest that both introns could have been acquired in a single event.      

Suppression of food intake and body weight gain by naloxone in rats

The effect of acute and chronic administration of naloxone on food acquisition and weight gain in rats was studied in 3 experiments. One injection of a sparingly-soluble salt of naloxone in slow-release vehicle markedly lowered mean food intake over that of control rats injected with the vehicle only. Mean body weight of the naloxone-injected rats was significantly lower than that of the control group for one week.Repeated evening injections (2000 h) of naloxone hydrochloride in saline tended to reduce the night-time feeding below control levels throughout the 10-day period of naloxone administration. Food intake was significantly lower in the 4- and 8-h periods after the first injection of naloxone than that on the preceding saline control night. The initial decreases were offset by increased day-time feeding so that total daily food intake was not significantly altered over the 10 days. When saline was substituted for naloxone, food intake increased.Rats given naloxone following 24 h of fasting consumed significantly less food and gained less weight during 4 h of access to food compared to those receiving saline. After a 48-h fast naloxone-treated rats also gained significantly less body weight than those given saline, but the reduction in food intake was not statistically significant. These results suggest the possibility that endorphins may have a modulating effect on feeding activity.     

Y chromosome variation of mice and men

DNA sequences from the nonrecombining portion of the Y chromosome were compared with autosomal and X-linked sequences from mice and humans to test the neutral prediction that ratios of polymorphism to divergence are the same for different genes. Intraspecific variation within Mus domesticus was compared with divergence between M. domesticus and Mus caroli for Sry, a region 5' to Sry, and four X-linked genes, Hprt, Plp, Amg, and Glra2. None of these comparisons revealed significantly reduced variation on the Y chromosome. Intraspecific variation within humans was compared with divergence between humans and chimpanzees for three Y-linked loci (Zfy, the YAP region, and the Sry region), seven X- linked loci (Il2rg, Plp, Hprt, Gk, Ids, Pdhal, and Dmd), and the beta- globin locus on chromosome 11. In these comparisons, the observed level of variation on the human Y chromosome was slightly lower than expected, but was significantly lower in only one case (Sry region vs. Dmd). These results suggest that the levels of variability on the Y chromosome in mice and humans are close to expected values given the effective population size and mutation rates for these loci. There is at most only a modest reduction in variability that may be attributed to natural selection (either genetic hitchhiking or background selection).      

Disintegration of adhesion plaques in chicken embryo fibroblasts upon Rous sarcoma virus-induced transformation: different dissociation rates for talin and vinculin

The localization of talin and vinculin in chicken embryo fibroblasts (CEF) during transformation was studied by immunoelectron microscopy. CEF cells were infected with a temperature-sensitive mutant of Rous sarcoma virus. After 16 h at 42 degrees C, transformation was induced by incubation at 37 degrees C for different intervals up to 3 h. Cells were cleaved by "wet cleaving" as reported previously by us (R. Brands and C.A. Feltkamp, 1988, Exp. Cell Res. 176, 309) and labeled with affinity-purified polyclonal antibodies to talin or vinculin, or monoclonal anti-vinculin. We observed a rapid reduction of vinculin in adhesion plaques within 15 min and a much slower dissociation of talin. This was found using single-labeling procedures and also within the same cell using double labeling. Seemingly intact microfilament bundles were observed associated with adhesion plaques that contained relatively little vinculin. These observations show that an early event in src-induced transformation is the release of vinculin from adhesion plaques. Furthermore, since adhesion plaques with attached filament bundles can exist at least transiently with very little or no vinculin present, it seems likely that vinculin is not, or not the only protein, linking actin filaments to adhesion plaques.     

Sister chromatid cohesion role for CDC28-CDK in Saccharomyces cerevisiae

High-fidelity chromosome segregation requires that the sister chromatids produced during S phase also become paired during S phase. Ctf7p (Eco1p) is required to establish sister chromatid pairing specifically during DNA replication. However, Ctf7p also becomes active during G(2)/M in response to DNA damage. Ctf7p is a phosphoprotein and an in vitro target of Cdc28p cyclin-dependent kinase (CDK), suggesting one possible mechanism for regulating the essential function of Ctf7p. Here, we report a novel synthetic lethal interaction between ctf7 and cdc28. However, neither elevated CDC28 levels nor CDC28 Cak1p-bypass alleles rescue ctf7 cell phenotypes. Moreover, cells expressing Ctf7p mutated at all full- and partial-consensus CDK-phosphorylation sites exhibit robust cell growth. These and other results reveal that Ctf7p regulation is more complicated than previously envisioned and suggest that CDK acts in sister chromatid cohesion parallel to Ctf7p reactions.     

Animal regeneration: ancestral character or evolutionary novelty?

An old question about regeneration is whether it is an ancestral character which is a general property of living matter, or whether it represents a set of specific adaptations to the different circumstances faced by different types of animal. In this review, some recent results on regeneration are assessed to see if they can throw any new light on this question. Evidence in favour of an ancestral character comes from the role of Wnt and bone morphogenetic protein signalling in controlling the pattern of whole‐body regeneration in acoels, which are a basal group of bilaterian animals. On the other hand, there is some evidence for adaptive acquisition or maintenance of the regeneration of appendages based on the occurrence of severe non‐lethal predation, the existence of some novel genes in regenerating organisms, and differences at the molecular level between apparently similar forms of regeneration. It is tentatively concluded that whole‐body regeneration is an ancestral character although has been lost from most animal lineages. Appendage regeneration is more likely to represent a derived character resulting from many specific adaptations.