Diversity of sequence haplotypes associated with beta-thalassaemia mutations in Algeria: implications for their origin.

We report the allelic sequence polymorphism associated with seven beta-thalassaemia mutations. Thirty-two DNAs originating from Algeria and 12 DNAs from Sardinia and Sicily were investigated. Their analysis revealed an association with a unique haplotype for three beta-thalassaemia mutations (-29, IVS-I-2 and IVS-I-1). It seems clear that these mutations have a unicentric origin. The presence of the -29 mutation could be explained by migration and founding effect. However, the local origin of IVS-I-2 seems clear. The four other mutations, FS6, IVS-I-6, IVS-I-110 and stop39 were found to be associated with at least two different sequence haplotypes. The likelihood of so many recurrent nucleotide dimorphisms in different lineages as a consequence of random mutation is very low; it is supported neither by the analysis of equivalent regions in other primates, nor by the presence of highly mutable sites such as CpG dinucleotides. The fact that these mutations are found exclusively in the Mediterranean area is not in favour of a recurrent origin of the mutation. The diversity is far more important for the preponderant thalassaemia mutations of the Mediterranean area and is higher in the 5' part of the beta-globin gene. Hence, the IVS-I-110, the preponderant beta-thalassaemia in the Eastern Mediterranean, probably emerged in the extension of the fertile crescent. For the stop39, all the data support the hypothesis of a West-Mediterranean origin. The diversity of haplotypes would then be generated by recombination events (crossing-over or gene conversions) between the original beta-thalassaemia chromosome and the other chomosomal structures present in the normal population.

Defective retroviral endogenous RNA is efficiently transmitted by infectious particles produced on an avian retroviral vector packaging cell line.

This report describes the contamination of "helper-free" stocks of defective retroviral vector with particles bearing retroviral endogenous RNA. An avian leukosis virus-based packaging cell line was developed from LMH cells that bear the ev1, ev3, and ev6 retroviral endogenous loci. The results show that an endogenous retroviral transcript (ev3) was packaged into virions produced by this packaging cell line and was efficiently transferred along with the vector to target cells. The titer of the ev contaminant particles was estimated at 50-100 CA-p27gag-expressing units/ml of supernatant.

Structure and expression of endogenous retroviral sequences in the permanent LMH chicken cell line.

From DNA mapping data, four endogenous proviral loci have been observed in the chicken permanent cell line LMH. The locus corresponding to endogenous virus (ev) ev1 is present in duplicate whereas the locus corresponding to ev3 is present in one copy. The other loci are probably ev6 and a solitary long terminal repeat. A RNA Northern blot analysis revealed both ev3 and ev6 transcripts but no ev1 transcript was detected. Using avian leukosis virus (ALV)-based vectors, transcomplementing assays were performed. They demonstrate the correct expression and maturation of endogenous env proteins and the absence of production of functional gag and pol components, indicating that these cells are not competent for viral production.

Use of retroviral vectors to introduce and express the beta-galactosidase marker gene in cultured chicken primordial germ cells.

Three methods of isolating primordial germ cells (PGCs) from gonads of 5-day-old chick embryos were compared. PGCs were then cultured in vitro in DMEM/F12 medium containing 10% fetal calf serum. BrdU incorporation showed that at least 10% of the PGC population were dividing, under our culture conditions, during the 2nd day of in vitro culture. During this culture period, PGCs were exposed to avian leukosis sarcoma virus-based retroviral vector pseudotyped with subgroup A envelope, carrying the LacZ reporter gene. X-Gal staining showed that PGCs were permissive to infection, with more than 50% of PGCs expressing the beta-Gal protein. These data represent the first demonstration that PGCs, isolated from gonads of 5-day-old chick embryos, are able to divide in vitro and that it is possible to introduce and express exogenous DNA in chick PGCs maintained in vitro.

Use of helper cells with two host ranges to generate high-titer retroviral vectors.

We have recently described Avian Leukosis Virus (ALV)-based packaging cell lines that can produce helper-free ALV-based retrovirus vectors with A, B, C, and E envelope host ranges. Here, we report that lacZ retroviral vectors of subgroup C or E can infect helper cells of subgroup A (Isolde) which are then able to produce high titers of lacZ recombinant viruses of subgroup A. Superinfection of helper cells by lacZ recombinant virus was performed by cocultivating packaging cells with two subgroup specificities (A and E), but this did not result in increased recombinant virus titers. This "ping-pong" process caused the emergence of replication-competent (RC) viruses which are shown to result from recombination between the viral sequences of the helper cell lines and the cis-acting sequences of the lacZ recombinant virus.

Packaging cells for avian leukosis virus-based vectors with various host ranges.

Using our previously described Haydée semipackaging cell line (F. L. Cosset, C. Legras, Y. Chebloune, P. Savatier, P. Thoraval, J. L. Thomas, J. Samarut, V. M. Nigon, and G. Verdier, J. Virol. 64:1070-1078, 1990) which produces avian leukosis virus gag and pol proteins, we have constructed packaging cells with subgroups B, C, and E envelope specificities. This allows us to produce helper-free avian leukosis virus particles carrying the lacZ reporter gene and the A, B, C, or E subgroup specificities. Titers of the recombinant lacZ virus are shown to be dependent upon the type of the env subgroup and the target avian cell.

In situ expression of helper-free avian leukosis virus (ALV)-based retrovirus vectors in early chick embryos.

Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.

Evolution of the primate beta-globin gene region: nucleotide sequence of the delta-beta-globin intergenic region of gorilla and phylogenetic relationships between African apes and man.

A 6.0-kb DNA fragment from Gorilla gorilla including the 5' part of the beta-globin gene and about 4.5 kb of its upstream flanking region was cloned and sequenced. The sequence was compared to the human, chimpanzee, and macaque delta-beta intergenic region. This analysis reveals four tandemly repeated sequences (RS), at the same location in the four species, showing a variable number of repeats generating both intraspecific (polymorphism) and interspecific variability. These tandem arrays delimit five regions of unique sequence called IG for intergenic. The divergence for these IG sequences is 1.85 +/- 0.22% between human and gorilla, which is not significantly different from the value estimated in the same region between chimpanzee and human (1.62 +/- 0.21%). The CpG and TpA dinucleotides are avoided. CpGs evolve faster than other sequence sites but do not confuse phylogenetic inferences by producing parallel mutations in different lineages. About 75% of CpG doublets have become TpG or CpA since the common ancestor, in agreement with the methylation/deamination pattern. Comparison of this intergenic region gives information on branching order within Hominoidea. Parsimony and distance-based methods when applied to the delta-beta intergenic region provide evidence (although not statistically significant) that human and chimpanzee are more closely related to each other than to gorilla. CpG sites are indeed rich in information by carrying substitutions along the short internal branch. Combining these results with those on the psi eta-delta intergenic region, shows in a statistically significant way that chimpanzee is the closest relative of human.

Newcastle disease virus (NDV) vaccine based on immunization with avian cells expressing the NDV hemagglutinin-neuraminidase glycoprotein.

Newcastle disease virus (NDV) is a paramyxovirus that bears two envelope glycoproteins at the virion surface. These proteins, fusion and hemagglutinin-neuraminidase (HN), are involved in the immune response against NDV infection. Recombinant cells constitutively expressing at their surface the HN protein from the velogenic Texas strain were generated by introducing the HN gene with a helper-free AEV-based vector. These recombinant cells were used to immunize chickens by various protocols, and birds were subsequently challenged with a lethal NDV injection. Both NDV protection and serologic response were observed.

Immune response and resistance to Rous sarcoma virus challenge of chickens immunized with cell-associated glycoproteins provided with a recombinant avian leukosis virus.

The Rous-associated virus 1 env gene, which encodes the envelope gp85 and gp37 glycoproteins, was isolated and inserted in place of the v-erbB oncogene into an avian erythroblastosis virus-based vector, carrying the neo resistance gene substituted for the v-erbA oncogene, to generate the pNEA recombinant vector. A helper-free virus stock of the pNEA vector was produced on an avian transcomplementing cell line and used to infect primary chicken embryo fibroblasts (CEFs) or quail QT6 cells. These infected cells, selected with G418 (CEF/NEA and QT6/NEA, respectively) were found to be resistant to superinfections with subgroup A retroviruses. The CEF/NEA preparations were used as a cell-associated antigen to inoculate adult chickens by the intravenous route compared with direct inoculations of NEA recombinant helper-free virus used as a cell-free antigen. Chickens injected with the cell-associated antigen (CEF/NEA) exhibited an immune response demonstrated by induction of high titers of neutralizing antibodies and were found to be protected against tumor production after Rous sarcoma virus A challenge. Conversely, no immune response and no protection against Rous sarcoma virus A challenge were observed in chickens directly inoculated with cell-free NEA recombinant virus or in sham-inoculated chickens.

Identification and structure analysis of endogenous proviral sequences in a Brown Leghorn chicken strain.

In a Brown Leghorn chicken strain, four endogenous proviral loci have been identified. The DNA mapping data show strong homology between their structures and that of the Rous-associated virus O (RAV-O) genome. Two of them seem similar to ev3 and ev6 loci previously described in White Leghorn chickens; the two others are unknown in White Leghorns. Using DNA amplification methods, envelope genes of these endogenous viral structures have been partially sequenced. The results demonstrate that subgroup-specific sequences of the endogenous loci were largely homologous with those of RAV-O.

Improvement of avian leukosis virus (ALV)-based retrovirus vectors by using different cis-acting sequences from ALVs.

Production and expression of double-expression vectors which transduce both Neo(r) and lacZ genes and are based on the structure of avian leukosis virus were enhanced by using cis-acting sequences (long terminal repeats and noncoding sequences) from Rous-associated virus-1 and Rous-associated virus-2 rather than those of avian erythroblastosis virus previously used in our constructs. Polyclonal producer cells obtained after transfection of these vectors into the Isolde packaging cell line gave rise to titers as high as 3 x 10(5) lacZ CFU/ml, whereas it was possible to isolate clones of producer cells giving rise to titers of more than 10(6) resistance focus-forming units per ml.

Origin and spread of beta-globin gene mutations in India, Africa, and Mediterranea: analysis of the 5' flanking and intragenic sequences of beta S and beta C genes.

Nucleotide polymorphisms of both the 5' flanking and intragenic regions of the human beta-globin gene were investigated by directly sequencing genomic DNA after amplification by the polymerase chain reaction in 47 subjects homozygous for the beta S or the beta C mutation. The sickle-cell mutation was found in the context of five different haplotypes defined by eight nucleotide substitutions and various structures of a region of the simple repeated sequence (AT) chi Ty. All subjects from the same geographic origin bear an identical chromosomal structure, defining the Senegal-, Bantu-, Benin-, Cameroon-, and Indian-type chromosomes. These results strengthen our previous conclusions about the multiple occurrence of the sickle-cell mutation. The Benin-type chromosome was also found among Algerian and Sicilian sickle-cell patients, whereas the Indian-type chromosome was observed in two geographically distant tribes, illustrating the spread of these sickle-cell genes. We also found that the intragenic sequence polymorphisms (frameworks) are not always in linkage disequilibrium with the BamH I polymorphism downstream from the beta-globin gene, as had been previously observed. Finally, we present a tentative phylogenetic tree of the different alleles at this locus. Some polymorphisms of this sequence might be contemporary with our last common ancestor, the great apes, that is, about 4-6 millions years old.

A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes.

An avian leukosis virus-based packaging cell line was constructed from the genome of the Rous-associated virus type 1. The gag, pol, and env genes were separated on two different plasmids; the packaging signal and the 3' long terminal repeat were removed. On a plasmid expressing the gag and pol genes, the env gene was replaced by the hygromycin resistance gene. The phleomycin resistance gene was inserted in the place of the gag-pol genes on a plasmid expressing the env gene. The plasmid containing the gag, pol, and Hygror genes was transfected into QT6 cells. Clones that produced high levels of p27gag were transfected with the plasmid containing the Phleor and env genes. Clones that produced high levels of env protein (as measured by an interference assay) were tested for their ability to package NeoR-expressing replication-defective vectors (TXN3'). One of the clones (Isolde) was able to transfer the Neo+ phenotype to recipient cells at a titer of 10(5) resistance focus-forming units per ml. Titers of supernatants of cells infected with Rous-associated virus type 1 prior to transfection by Neor vectors were similar. Tests for recombination events that might result in intact helper virus showed no evidence for the generation of replication-competent virus. The use of selectable genes inserted next to the viral genes to generate high-producer packaging cell lines is discussed.

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.

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

We 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.

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

A 5600-base-pair (bp) fragment including the beta-globin gene and about 4000 bp of its 5' flanking sequence was cloned from the DNA of Macaca cynomolgus (an Old World monkey), and the 5' flanking region was sequenced. Comparison with human, chimpanzee, mouse, rabbit, and Xenopus 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 pseudo-eta-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 X 10(-9) substitution/site/year.

Nucleotide sequence of the beta-globin genes in gorilla and macaque: the origin of nucleotide polymorphisms in human.

Part of the beta-globin genes of Macaca cynomolgus and Gorilla 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.

Evolution of the primate beta-globin gene region. High rate of variation in CpG dinucleotides and in short repeated sequences between man and chimpanzee.

A 5500 base-pair fragment including the beta-globin gene downstream from codon 122 and about 4000 base-pairs of its 5' flanking sequence was cloned from chimpanzee DNA and thoroughly sequenced before being compared with the corresponding human sequence: 88 point differences (83 substitutions and 5 deletions or insertions of 1 base-pair) were detected as well as seven more important deletion/insertion events. These changes occur preferentially in two kinds of structure. First, 40% of the CpG dinucleotides present in either human or chimpanzee sequences are affected by nucleotide variations. This corresponds to a divergence level considerably higher than that expected. Second, most short repeated sequences found in the 5' extragenic sequence are involved in mutational events (amplification or contraction of the number of basic motifs as well as point substitutions or deletions/insertions of 1 base-pair). Considering the very low level of nucleotide sequence divergence between these two closely related species, our data provide direct evidence for CpG and tandem array instability.

A new method for detection of small modifications in genomic DNA, applied to the human delta-beta globin gene cluster.

Cloned DNA fragments were subcloned in filamentous coliphages fd 103 or M 13; the recombinant single-stranded DNAs were then used to form hybrids with genomic DNA as well as with complementary recombinant single-stranded DNA. Hybrids were submitted to S1-nuclease treatment alone or in combination with restriction enzyme digestions. This method was used to analyze the delta-beta globin gene cluster from the total genomic DNA of a beta 0-thalassemic patient. A modification located approximately 530 base pairs upstream from the cap site of the beta-globin gene was detected in only one thalassemic chromosome of this patient. Sequence analysis have shown that the patient was homozygous for a single nucleoside change (dC----dT) which remains undetected by our hybridization method, leading to a codon 39 nonsense mutation; they have demonstrated too that he was heterozygous for the modification mentioned and detected by S1-nuclease, which corresponds to an additional sequence d(T-A-T-A) in a 52 alternating purine-pyrimidine run, leading to a complex change from d[(A-T)7(T)7] to d[(A-T)11(T)3].