Prenatal and adult androgen activities in alcohol dependence.

OBJECTIVE: Alcohol dependence is more prevalent in men than in women. The evidence for how prenatal and adult androgens influence alcohol dependence is limited. We investigated the effects of prenatal and adult androgen activity on alcohol dependence. Moreover, we studied how the behaviours of pregnant women affect their children's prenatal androgen load. METHOD: We quantified prenatal androgen markers (e.g., second-to-fourth finger length ratio [2D : 4D]) and blood androgens in 200 early-abstinent alcohol-dependent in-patients and 240 controls (2013-2015, including a 12-month follow-up). We also surveyed 134 women during pregnancy (2005-2007) and measured the 2D : 4D of their children (2013-2016). RESULTS: The prenatal androgen loads were higher in the male alcohol-dependent patients compared to the controls (lower 2D : 4D, P = 0.004) and correlated positively with the patients' liver transaminase activities (P < 0.001) and alcohol withdrawal severity (P = 0.019). Higher prenatal androgen loads and increasing androgen levels during withdrawal predicted earlier and more frequent 12-month hospital readmission in alcohol-dependent patients (P < 0.005). Moreover, stress levels (P = 0.002), alcohol (P = 0.010) and tobacco consumption (P = 0.017), and lifetime stressors (P = 0.019) of women during pregnancy related positively to their children's prenatal androgen loads (lower 2D : 4D). CONCLUSION: Androgen activities in alcohol-dependent patients and behaviours of pregnant women represent novel preventive and therapeutic targets of alcohol dependence.

Field anaesthesia of leatherback sea turtles (Dermochelys coriacea).

Ten nesting leatherback sea turtles on Trinidad were anaesthetised for electroretinogram (ERG) measurements, using ketamine and medetomidine, reversed with atipamezole. They weighed 242 to 324 kg and were given initial doses of 3 to 8 mg/kg ketamine and 30 to 80 microg/kg medetomidine administered into an external jugular vein; six of the turtles received supplementary doses of 2.6 to 3.9 mg/kg ketamine combined with 0 to 39 microg/kg medetomidine. The lower doses were used initially to ensure against overdosage and reduce the chances of residual effects after the turtles returned to the water, but successful ergs called for step-wise dose increases to the required level of anaesthesia. Respiratory rate, heart rate, electrocardiogram, cloacal temperature, and venous blood gases were monitored, and blood was collected for plasma biochemistry. At the end of the erg procedure, atipamezole was administered at 150 to 420 microg/kg (five times the dose of medetomidine), half intramuscularly and half intravascularly. The turtles were monitored and prevented from re-entering the water until their behaviour was normal. No apparent mortalities or serious anaesthetic complications occurred. The observed within-season return nesting rate of the anaesthetised turtles was comparable with that of unanaesthetised turtles.

Treatment with DNA-damaging agents increases expression of polA'-'lacZ gene fusions in Escherichia coli K-12.

The polA gene of Escherichia coli encodes the DNA polymerase I that is involved in DNA replication and repair. In contrast to the extensive body of data on the structure and function of polymerase I, there is little information available concerning the mechanisms that govern polA expression. Here, we studied the expression of the polA gene using translational fusions to lacZ. We found that treatment with the DNA-damaging agents 4-nitroquinoline-N-oxide (4-NQO), UV light mitomycin C (MC) and methyl methanesulfonate (MMS) leads to enhanced expression of polA'-'lacZ fusions. The increase in expression of polA reflects stimulation of transcription from a single promoter, as determined by S1 nuclease analyses. This was not observed in mutants that are blocked in induction of the SOS regulon. However, mutants with defective excision repair were more susceptible to polA stimulation. These results support the hypothesis that increased polA expression may be important for the ability to repair bulky DNA adducts that interfere with replication.

The role of plastidial transporters in developing embryos of oilseed rape (Brassica napus L.) for fatty acid synthesis.

The phosphoenolpyruvate transporter (PPT) is one of several important transporters for channelling carbon intermediates utilized for fatty acid synthesis and other plastidial pathways from the cytosol into the plastid. In this paper we show results on how the activity of the PPT changes between two important, physiologically different developmental stages of oilseed rape embryos.

A family of differentially amplified repetitive DNA sequences in the genus Beta reveals genetic variation in Beta vulgaris subspecies and cultivars.

Members of a highly abundant restriction satellite family have been isolated from the wild beet species Beta nana. The satellite DNA sequence is characterized by a conserved RsaI restriction site and is present in three of four sections of the genus Beta, namely Nanae, Corollinae, and Beta. It was not detected in species of the evolutionary old section Procumbentes, suggesting its amplification after separation of this section. Sequences of eight monomers were aligned revealing a size variation from 209 to 233 bp and an AT content ranging from 56.5% to 60.5%. The similarity between monomers in B. nana varied from 77.7% to 92.2%. Diverged subfamilies were identified by sequence analysis and Southern hybridization. A comparative study of this repetitive DNA element by fluorescent in situ hybridization and Southern analyses in three representative species was performed showing a variable genomic organization and heterogeneous localizations along metaphase chromosomes both within and between species. In B. nana the copy number of this satellite, with some 30,000 per haploid genome, is more than tenfold higher than in Beta lomatogona and up to 200 times higher than in Beta vulgaris, indicating different levels of sequence amplification during evolution in the genus Beta. In sugar beet (B. vulgaris), the large-scale organization of this tandem repeat was examined by pulsed-field gel electrophoresis. Southern hybridization to genomic DNA digested with DraI demonstrated that satellite arrays are located in AT-rich regions and the tandem repeat is a useful probe for the detection of genetic variation in closely related B. vulgaris cultivars, accessions, and subspecies.

Analysis and chromosomal localization of retrotransposons in sugar beet (Beta vulgaris L.): LINEs and Ty1-copia-like elements as major components of the genome.

DNA sequences of the reverse transcriptase gene of long terminal repeat (LTR) and non-LTR (non-viral) retrotransposons have been isolated and cloned from the genome of sugar beet (Beta vulgaris). Both retrotransposon types are highly amplified in sugar beet and may account for 2-5% of the genome. The BNR1 family, representing the first non-viral retrotransposon reported from a dicotyledonous species, shows homology to the mammalian L1 family of long interspersed repeated sequences (LINEs) and to retrotransposable elements from maize and lily. Sequences of the Tbv family are homologous to the Ty1-copia class of LTR retrotransposons. The BNR1 and Tbv retrotransposon families are characterized by sequence heterogeneity and are probably defective. The deduced peptide sequences were used to investigate the relation to other retroelements from plants, insects and mammals. Fluorescence in situ hybridization was used to investigate the physical distribution and revealed that both retrotransposon families are present on all sugar beet chromosomes and largely excluded from chromosomal regions harbouring the 18S-5.8S-25S rRNA genes. The BNR1 family is organized in discrete clusters, while the Tbv family of Ty1-copia-like retrotransposons shows a more uniform distribution along chromosome arms and is absent from some chromosomal regions. These contrasting distributions emphasize the differences in evolutionary amplification and dispersion mechanisms between the two types of retrotransposons. The in situ results of both elements reflect significant features of a higher order structure of the genome, as it is known for both short interspersed repeated sequences (SINEs) and LINEs in human.

The genomic organization of non-LTR retrotransposons (LINEs) from three Beta species and five other angiosperms.

We have isolated and characterized conserved regions of the reverse transcriptase gene from non-LTR retrotransposons, also called long interspersed nuclear elements (LINEs), from Beta vulgaris, B. lomatogona and B. nana. The novel elements show strong homology to other non-LTR retrotransposons from plants, man and animals. LINEs are present in all species of the genus Beta tested, but there was variation in copy number. Analysis by Southern hybridization and fluorescent in situ hybridization revealed the clustered organization of these retroelements in beet species. PCR amplification using degenerate primers to conserved motifs of the predicted LINE protein sequence enabled the cloning of LINEs from both Monocotyledonae (Allium cepa, Oryza sativa and Secale cereale) and Dicotyledonae (Nicotiana tabacum and Antirrhinum majus) indicating that LINEs are a universal feature of plant genomes. A dendrogram of fifteen new and six previously isolated sequences showed the high level of sequence divergence while revealing families characteristic of some genera. The genomic organization of non-LTR retrotransposons was examined more detailed in A. majus and O. sativa.

Comparative analysis of the chromosomal and genomic organization of Ty1-copia-like retrotransposons in pteridophytes, gymnosperms and angiosperms.

We have investigated the physical distribution of the reverse transcriptase genes of Ty1-copia-like retrotransposable elements from 12 plant species belonging to different subdivisions by hybridization in situ on chromosome preparations. Ty1-copia-like elements showed different and non-random hybridization patterns. A dispersed distribution throughout most of the chromosomes with reduced hybridization at some regions or with some weak clustering at other regions was found in Allium cepa, Beta vulgaris, Brassica campestris, Brassica oleracea, Pennisetum glaucum, Pinus elliottii, Selaginella apoda, Vicia faba and Vicia narbonensis. Reduced hybridization occurred mainly at centromeric regions, nucleolus-organizing regions and regions known to be mainly composed of tandemly repeated sequences. In the fern Pteris cretica the retroelements showed a dispersed genomic organization with clustering at some chromosomal regions and whole chromosomes showing little signal. In Arabidopsis thaliana and Cicer arietinum Ty1-copia-like elements were found in clusters at the paracentromeric heterochromatin, a novel organization for a repetitive element in A. thaliana. New retroelement families were isolated from A. thaliana and from Beta vulgaris. Alignment of the deduced peptide sequences with Ty1-copia-like elements from other plants showed considerable divergence which was used to calculate their relationships, indicating the value of reverse transcriptase gene analysis in phylogenetic and biodiversity studies.

The chromosomal distributions of Ty1-copia group retrotransposable elements in higher plants and their implications for genome evolution.

Retrotransposons make up a major fraction--sometimes more than 40%--of all plant genomes investigated so far. We have isolated the reverse transcriptase domains of the Ty1-copia group elements from several species, ranging in genome size from some 100 Mbp to 23,000 Mbp, and determined the distribution patterns of these retrotransposons on metaphase chromosomes and within interphase nuclei by DNA:DNA in situ hybridization. With some exceptions, the reverse transcriptase domains were distributed over the length of the chromosomes. Exclusion from rDNA sites and some centromeres (e.g., slash pine, 23,000 Mbp, or barley, 5500 Mbp) is frequent, whereas many species exclude retrotransposons from other sites of heterochromatin (e.g., intercalary and centromeric sites in broad bean). In contrast, in the plant Arabidopsis thaliana, widely used for plant molecular genetic studies because of its small genome (c. 100 Mbp), the Ty1-copia group reverse transcriptase gene domains are concentrated in the centromeric regions, colocalizing with the 180 bp satellite sequence pAL1. Unlike the pAL1 sequence, however, the Ty1-copia signal is also detectable as weaker, diffuse hybridization along the lengths of the chromosomes. Possible mechanisms for evolution of the contrasting distributions are discussed. Understanding the physical distribution of retrotransposons and comparisons of the distribution between species is critical to understanding their evolution and the significance for generation of the new patterns of variability and in speciation.