Interspersion of different repeated sequences in the wheat genome revealed by interspecies DNA/DNA hybridisation

The repeated sequences in oats DNA have been used to study chromosomal repeated sequence organisation in wheat. Approximately 75% of the wheat genome consists of repeated sequences but only approximately 20% will form heteroduplexes with repeated sequences from oats DNA at 60 degrees C in 0.18 M Na+. The proportion of wheat DNA that forms heteroduplexes with oats DNA is shown to be independent of the wheat DNA fragment length. However, the proportion of wheat DNA that is retained with the heteroduplexes when fractionated on hydroxyapatite is very dependent upon the wheat fragment length up to 3500 nucleotides. This is because more non-renatured wheat DNA is attached to the heteroduplexes with longer fragments. The results indicate that the repeated sequences in the wheat genome homologous to repeated sequences in oats are not clustered in the chromosomes but distributed amongst other repeated and possible non-repeated sequences.     

Inverted repetitive sequences in the human genome

A specific class of DNA sequences, the inverted repetitive sequences, forms hairpin-like structures in denatured DNA by the folding back of a single linear chain. The reassociation process of these sequences is unimolecular and the rate is extremely fast. Inverted repetitive sequences comprise 6% of the total human genome. They appear to be heterogeneous in length with an overall average length of 190 nucleotides. The inverted sequences are represented in almost all families of repetition frequencies, highly repetitive as well as very few copies per genome. They are not localized at unique sites on metaphase chromosomes. It is estimated that there are approximately 2 X 10(6) inverted repeats per haploid human genome. The biological function of this class of sequences is unknown.     

Endogenous retroviral sequences present in the human genome

The common carotid arteries of normal adult rats were investigated electron-microscopically after tannic acid fixation. This fixation technique yields a better demonstrability of the structures of the connective tissue, the basal laminae and the surface coat of the cell membrane. The common carotid artery represents a vessel of the elastic type. The intima consists of an endothelium and a narrow gap of connective tissue (0.1-1 micron) which contains single collagenous fibrils and small elastic structures. This space is only occasionally as wide as 3 microns, especially beneath gaps of the internal elastic membrane. In these areas, single cells and structures of densely packed filaments are additionally observed which can neither be attributed to collagenous fibrils nor to elastic fibres. The intima is demarcated from the outside by an internal elastic membrane (1 micron) which shows a number of gaps. The media exhibits 3 to 4 elastic membranes without gaps. Smooth muscle cells of the contractile type stretch in an oblique direction between these membranes, i.e. they are not arranged in a circular or spiral manner. Most of their process-rich ends are inserted directly into the elastic material and not via a basal lamina. Processes from these smooth muscle cells, collagenous fibrils and elastic fibres are seen in the intercellular spaces. The muscle cells are occasionally interlinked by gap junctions. The basal lamina does not surround the muscle cells continuously. The adventitia contains bundles of collagenous fibrils, fibrocytes, a few small vessels and nerves with a perineuronal envelope. Nerves could not be demonstrated in the media. The oblique course of the smooth muscle cells and the insertion into the elastic membranes indicate that these cells do not predominantly contribute to changes in the width of the lumen but also serve the stabilisation and resetting of the elastic membranes. Contraction is probably induced by an opening of stretch-dependent Ca2+ channels. Due to the interlinkage with gap junctions, the muscle cells of one layer respond as a functional unit. Our findings provide a morphological basis for elucidating commonly encountered changes, such as smooth muscle migration through a normally interrupted inner elastic lamina.     

The genome organization of the broad bean necrosis virus (BBNV)

In this study the effect of lipoprotein lipase (LPL) on the selective uptake of high density lipoprotein (HDL) cholesteryl esters (CE) by hepatic cells was investigated. Human HDL3 (d 1.125-1.21 g/ml) was radiolabeled with 125I in the protein moiety and with 3H in the CE moiety. LPL was prepared from bovine milk. Human hepatocytes in primary culture and human Hep3B hepatoma cells were incubated in medium containing doubly radiolabeled HDL3 with or without LPL. Without LPL, apparent HDL3 particle uptake according to the lipid tracer (3H) was in excess of that due to the protein label (125I) indicating selective CE uptake from HDL3. Addition of LPL increased selective CE uptake up to 7-fold. This stimulation of HDL3 selective CE uptake was independent of the lipolytic activity of LPL as suggested by several experimental approaches. Cell surface heparan sulfate proteoglycan deficiency decreased the LPL-mediated increase in selective CE uptake suggesting an important role for these molecules. In low density lipoprotein (LDL) receptor- or LDL receptor-related protein-(LRP)-deficient cells, LPL increased selective CE uptake as it did in normal cells yielding no evidence that these receptors play a role in the LPL effect on selective CE uptake. In summary, lipoprotein lipase increases the selective uptake of high density lipoprotein-associated cholesteryl ester by hepatic cells in culture. This effect is dependent on cell surface heparan sulfate proteoglycans but independent of lipolysis and of endocytosis mediated by low density lipoprotein receptor-related or low density lipoprotein receptors.     

Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome

Two adeno-associated virus (AAV) elements are necessary for the integration of the AAV genome: Rep78/68 proteins and inverted terminal repeats (ITRs). To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration efficiencies of three different plasmids containing a green fluorescent protein (GFP) expression cassette. In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter gene (integration cassette), and a third plasmid consisted of an integration cassette plus a Rep78 expression cassette. One day after transfection of 293 cells, fluorescent cells were sorted by flow cytometry and plated at 1 cell per well. Two weeks after sorting, colonies were monitored for stable expression of GFP. Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent colonies. Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid containing both the integration cassette and Rep78 expression cassette produced stable fluorescent colonies at a frequency of 47% +/- 7.5%. Southern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVSI site in more than 50% of the clones analyzed. Some clones also showed tandem arrays of the integrated GFP cassette. Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78.     

The role of the paternal genome in the development of the mouse germ line

The mouse germ line originates at 6.5 days post coitum (dpc) in the proximal epiblast, apparently in response to signals from the primitive endoderm or the extraembryonic mesoderm [1,2]. Some studies have implied a significant role for imprinted genes in germ-line development [3,4]. These genes, whose expression is determined by their parental origin [5], serve complementary functions during mammalian development [6-9] and exert striking reciprocal phenotypic effects on androgenetic (AG: two paternal genomes) and parthenogenetic (GG/PG: two maternal genomes) cells [3,4,10]. This may include a fundamental effect on germ-cell development because PG but not AG cells can differentiate into viable gametes [3,4,11], suggesting that the maternal genome is obligatory for development of the mammalian germ line. Here we show unequivocally that AG cells can differentiate into germ cells, and that in chimeras with normal cells they produce functional sperm. These studies establish that the paternal and maternal genomes can individually provide both the signal and the response required for the specification of germ cells in mammals.     

Variation in genome organization of the plant pathogenic fungus Colletotrichum lindemuthianum

The effects of 5-HT3 receptor antagonists on ethanol intake were examined in the selectively bred alcohol-preferring P line of rats under continuous and limited access to 10% (v/v) ethanol with food and water ad lib. Single daily injections of either MDL 72222 (MDL) or ICS 205-930 (ICS) (0.01-3.0 mg/kg, SC) given 60 min before a 4-h scheduled access period for 4 consecutive days failed at all doses to alter the intake of a 10% (v/v) ethanol solution by P rats. However, multiple daily injections of either MDL (1-3 mg/kg, SC) or ICS (3.0 and 5.0 mg/kg, SC), given three times daily at 4-h intervals, significantly reduced ethanol intake under 24-h free-choice conditions on the first treatment day. Additionally, a single administration of 1.0 mg/kg MDL reduced 24-h free-choice ethanol intake by approximately 50% of control values and had no effect on 24-h saccharin intake. The effects of MDL were further examined in a 2-h schedule access paradigm in which rats received the access period at the same time every day (Fixed) or randomly during the dark cycle (Variable). Although 1.0 mg/kg MDL had little effect on ethanol drinking in the Fixed group, ethanol intake was reduced by 55% of control levels in the Variable group. Overall, the data indicate that drinking conditions influence the effectiveness of 5-HT3 antagonists to reduce ethanol consumption. Furthermore, the results suggest that conditions, associated with limited access ethanol drinking, markedly reduce the actions of 5-HT3 antagonists on ethanol intake.     

Genomic organization of the mouse adrenocorticotropin receptor

PURPOSE: This paper describes the perceived risk of occupationally contracting HIV and reported compliance with universal precaution guidelines among Australian dental hygienists and dentists. METHODS: This examination is based upon responses to a mailed questionnaire from all registered dental hygienists (63% response rate, n = 208) and dentists (76% response rate, n = 550) in Western Australia. RESULTS: Results indicate that: 1) oral healthcare providers who perceive a high risk of occupationally contracting HIV report a more conservative, cautious approach to HIV infection than do providers who perceive less risk of contracting the virus; 2) dental hygienists are more likely than dentists to report a higher degree of perceived risk of occupationally contracting HIV; and 3) dentists are more likely than dental hygienists to report compliance with universal precaution guidelines in the dental practices where they work. CONCLUSION: Educating oral healthcare providers on the realistic risks of occupationally contracting HIV and the value of compliance with universal precaution guidelines may reduce undue stress and hindrances in the provision of safe and effective oral healthcare in this era of AIDS.     

Ribosomal S27a coding sequences upstream of ubiquitin coding sequences in the genome of a pestivirus

Molecular characterization of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP Rit, a temperature-sensitive strain widely used for vaccination, revealed that the viral genomic RNA is about 15.2 kb long, which is about 2.9 kb longer than the one of noncytopathogenic (noncp) BVDV strains. Molecular cloning and nucleotide sequencing of parts of the genome resulted in the identification of a duplication of the genomic region encoding nonstructural proteins NS3, NS4A, and part of NS4B. In addition, a nonviral sequence was found directly upstream of the second copy of the NS3 gene. The 3' part of this inserted sequence encodes an N-terminally truncated ubiquitin monomer. This is remarkable since all described cp BVDV strains with ubiquitin coding sequences contain at least one complete ubiquitin monomer. The 5' region of the nonviral sequence did not show any homology to cellular sequences identified thus far in cp BVDV strains. Databank searches revealed that this second cellular insertion encodes part of ribosomal protein S27a. Further analyses included molecular cloning and nucleotide sequencing of the cellular recombination partner. Sequence comparisons strongly suggest that the S27a and the ubiquitin coding sequences found in the genome of CP Rit were both derived from a bovine mRNA encoding a hybrid protein with the structure NH2-ubiquitin-S27a-COOH. Polyprotein processing in the genomic region encoding the N-terminal part of NS4B, the two cellular insertions, and NS3 was studied by a transient-expression assay. The respective analyses showed that the S27a-derived polypeptide, together with the truncated ubiquitin, served as processing signal to yield NS3, whereas the truncated ubiquitin alone was not capable of mediating the cleavage. Since the expression of NS3 is strictly correlated with the cp phenotype of BVDV, the altered genome organization leading to expression of NS3 most probably represents the genetic basis of cytopathogenicity of CP Rit.