Gérard Verdier

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying theNeor selectable marker and theEscherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G1 progeny at a frequency of 2.7%.Neor andlacZ genes were transcribedin vitro in chicken embryo fibroblast cultures from transgenic embryos of the G2 progeny.

Nucleotide Sequence of the Beta-Globin Genes in Gorilla and Macaque: The Origin of Nucleotide Polymorphisms in Human

SummaryPart of the beta-globin genes ofMacaca cynomolgus andGorilla gorilla has been cloned and sequenced. Ten putatively neutral nucleotide polymorphisms have been described at the beta-globin locus in humans. They are associated in seven combinations, which define seven different haplotypes of the beta-globin gene: four major frameworks—1, 2, 3, and 3*—and three minor frameworks, which we term KI1, KA1, and OR1. The nucleotide sequences of these frameworks are compared with those of homologous sequences in chimpanzee, colobus, macaque, and gorilla. This comparison provides strong evidence that framework 2 was the earliest framework in the human lineage. From framework 2, a rooted parsimonious tree for the six other frameworks is constructed. This phylogenetic tree is discussed in terms of the evolution of nucleotide polymorphisms as well as in terms of genetic affinities between human populations.For each position at which there is base difference in comparing human, gorilla, and chimpanzee beta-globin genes, the phyletic lineage where the corresponding substitution occurred has been identified using the maximum parsimony procedure. The data provide evidence that polymorphisms may represent a significant component of differences between closely related species. If so, nucleotide polymorphisms may strongly bias estimates of small evolutionary distances.

Avian retroviral vectors derived from avian defective leukemia virus: Role of the translational context of the inserted gene on efficiency of the vectors

We have constructed retroviral vectors derived from the genome of avian erythroblastosis virus ES4 (AEV ES4). The neo selectable gene was substituted for the original v-erbA or v-erbB oncogenes of AEV, either in the same or in a different reading frames. Recombinant retrovirus were rescued and used to infect chicken embryo fibroblasts or quail QT6 cells. When the neo gene was inserted in the same reading frame as the original oncogene, we obtained (1) a high level of expression of the neo gene, (2) a balanced ration of both genomic and subgenomic RNAs, and (3) high titer recombinant viruses. Conversely, when the neo gene was inserted in a reading frame different from that of the original oncogene, we observed (1) a very low level of expression of the neo protein, (2) a predominance of the viral transcript used as translational template for the neo protein synthesis, and (3) low titer recombinant viruses. One of the vectors was used to transfer a human delta-globin gene into avian cells in culture without detectable rearrangement of this gene, but exhibited a deletion within the conserved noncoding region located between the two original oncogenes. Our data provide information for further construction of double expression vectors. Furthermore, three of the vectors would provide helpful tools to identify genetic elements of the virus genome involved in splicing regulation.

Nucleotide sequence of the delta-beta-globin intergenic segment in the macaque: Structure and evolutionary rates in higher primates

SummaryA 5600-base-pair (bp) fragment including the beta-globin gene and about 4000 bp of its 5′ flanking sequence was cloned from the DNA ofMacaca cynomolgus (an Old World monkey), and the 5′ flanking region was sequenced. Comparison with human, chimpanzee, mouse, rabbit, andXenopus orthologous sequences reveals a tandemly repeated sequence called RS4 at the same position (about 500 bp 5′ from the transcription start of the adult beta-globin gene) in all six species. We suggest that a tandemly repeated sequence has been maintained by functional constraints since the divergence between amphibians and reptiles.Excluding tandemly repeated sequences as well as about 400 nucleotides upstream from the cap site, the average base substitution frequencies among human, chimpanzee, and macaque intergenic sequences were calculated. They appear to be strongly correlated with the delta T50 values measured between the corresponding nuclear DNAs. They are also similar to base substitution frequencies calculated by Chang and Slightom (1984) at the pseudoeta-globin locus. Thus, exclusion of sequences involved in specific modes of variation might allow the use of intergenic sequences for the accurate calculation of genetic distances.Using a time scale based on the dating of the Atlantic split, we estimate the base substitution rate of primate noncoding DNA to be 1.0×10−9 substitution/site/year.

Recent insertion of an alu sequence in the beta-globin gene cluster of the gorilla

SummaryWe present the nucleotide sequence of a new Alu family member that lies between the delta- and beta-globin genes in gorilla DNA. The sequence exhibits 91% similarity with a consensus sequence of the Alu family. It is flanked by a perfect repetition of a 16-nucleotide target sequence and terminates with 24 adenylic residues. As this sequence is absent at this locus in other primate DNAs, its insertion occurred less than 8 million years ago, thus supporting the idea that Alu sequences are still mobile elements in the hominoid genome.

Synthesis and translation site of light-induced mRNAs in etiolated Euglena gracilis

Poly(a)-mRNA synthesis has been studied in etiolated Euglena gracilis exposed to 1 or 2 h of illumination. 1. Labeling kinetics of mRNAs containing poly(A) sequences, during illumination or after return to darkness, reach a plateau in 10 or 20 min according to nutritional conditions. When the cultures are returned to darkness, the mRNA synthesis decreases rapidly. Thus, the synthesis of these mRNAs (light-induced mRNAs) is dependent on light and their half-life can be evaluated. 2. Cycloheximide induces accumulation of label in poly(A)-containing mRNA; such an accumulation is not observed after addition of lincomycin. Labeling during illumination of mRNA in a chloroplast mutant is similar to that in the wild type strain. These data suggest that the poly(A)-mRNAs synthesized in the two first hours of illumination are translated on cytoplasmic polyribosomes.

Transposable elements behavior following viral genomic stress inDrosophila melanogaster inbred line

To analyze the behavior of endogenous transposable elements under genomic stress, aDrosophila melanogaster inbred line was submitted to three kinds of viral perturbations. First, a retroviral plasmid containing the avian Rous Associated Virus type 2 (RAV-2) previously deleted for the viral envelope coding gene (env) was introduced by P element transformation into theDrosophila genome. An insertion of this avian retroviral sequence was detected byin situ hybridization in site 53C on polytene chromosome arm 2R. Second,Drosophila embryos were injected with RAV-2 particles produced by cell culture after transfection with the retroviral plasmid. Third, theDrosophila melanogaster inbred line was stably infected by the sigma native virus. It appears that neither the offspring of the flies in which the viral DNA was found integrated nor those from the infected sigma flies showed copia or mdgl element mobilization. Injection of the avian RAV-2 particles led, however, to the observation of somatic transpositions of mdgl element on the 2L chromosome, the copia element insertion pattern remaining stable. Thus, endogenous transposable elements show more instability in sublines injected with exogenous viral particles than in a transgenic subline containing a foreign viral insert, all transposable elements not being equally sensitive to such genomic stress.

Tissue specificity of retrovirus expression in inoculated avian embryos revealed by in situ hybridization to whole-body section

To examine the mechanism of viral tropism in vivo, we injected RAV(1) avian retrovirus into 1-day-old chicken embryos. After 8 days, the majority of the embryos became infected. In situ hybridization to whole embryo sections revealed high levels of intracellular viral RNA, apparently restricted to skeletal muscle cells. The most likely interpretation is that expression of this virus is regulated at the transcriptional level by one or more tissue-specific endogenous factors.

CG sites and their nucleotide environment in the human γ-δ-β-globin gene cluster: distribution, frequency and possible frame for differential methylation

Resume o 1. On a cherche a detecter la position des dinucleotides CG dans le DNA genomique humain couvrant la region des genes de globines γ-δ-β. Lexistence et la repartition de ces dinucleotides sont, en effet, susceptibles dintervenir dans les potentialites de methylation de ces regions et dans les consequences de la methylation au niveau de lexpression des genes. 2. Apres clonage, les DNA concernes ont ete analyses par une batterie dendonucleases de restriction comportant un dinucleotide CG dans leur sequence de reconnaissance (sequences que nous appellerons «CG); la majorite de ces enzymes sont inactives lorsque la cytosine du dinucleotide CG est methylee. Les clones etant cultives dans Escherichia coli, seules les cytosines internes des sequences CCAT GG sont susceptibles detre methylees; ainsi, tous les sites contenant «CG restent donc reconnaissables par ces enzymes. 3. La frequence des sites «CG diminue regulierement depuis la region des genes γ jusqua la region du gene β, cette derniere ne comportant que tres peu de sites «CG. Lensemble des enzymes utilisees dans cette etude permet de deceler quatre fois plus de sites enzymatiques «CG que le nombre detecte par le seul emploi des isoschizomeres Msp I/Hpa II. 4. Letude des zones deja sequencees dans la region etudiee a montre que les dinucleotides CG (inclus ou non dans une sequence «CG) y sont repartis a raison de 1 pour 104 nucleotides au niveau des genes γ et 1 pour 138 nucleotides au niveau du gene β. Dans la region des genes γ, 20–25 per cent des dinucleotides CG sont integres dans des sequences «CG, cette valeur tombant a moins de 10 p. cent dans la region du gene β. 5. Lenvironnement nucleotidique des dinucleotides CG revele des situations particulieres comportant une majorite de 5′ … CGG … 3′ dans les regions 5′ adjacentes des genes γ et une majorite de 5′ … ACG … 3′ dans la region 5′ adjacente du gene β. 6. Lensemble des resultats suggere des considerations nouvelles concernant lanalyse de la methylation des cytosines susceptible detre reliee aux activites respectives des genes γ, δ et β.

Molecular cloning and sequencing of a cDNA encoding a β-thyroid hormone receptor in muscovy duckling

A cDNA clone encoding a beta-thyroid hormone receptor (TRbeta) from muscovy duckling liver was isolated and sequenced. Comparison with the chicken TRbeta sequence showed a high degree of homology. This cDNA was used as a probe to characterize the TRbeta mRNA transcripts expressed in muscovy duckling liver.