This study was carried out to elucidate whether primordial germ cells, obtained from embryonic blood and transferred into partially sterilized male and female recipient embryos, could differentiate into functional gametes and give rise to viable offspring. Manipulated embryos were cultured until hatching and the chicks were raised until maturity, when they were mated. When the sex of the donor primordial germ cells and the recipient embryo was the same, 15 out of 22 male chimaeric chickens (68.2%) and 10 out of 16 female chimaeric chickens (62.5%) produced donor-derived offspring. When the sex of the donor primordial germ cells and the recipient embryo was different, 4 out of 18 male chimaeric chickens (22.2%) and 2 out of 18 female chimaeric chickens (11.1%) produced donor-derived offspring. The rates of donor-derived offspring from the chimaeric chickens were 0.6-40.0% in male donor and male recipient and 0.4-34.9% in female donor and female recipient. However, the rates of donor-derived offspring from the chimaeric chickens were 0.4-0.9% in male donor and female recipient and 0.1-0.3% in female donor and male recipient. The presence of W chromosome-specific repeating sequences was detected in the sperm samples of male chimaeric chickens produced by transfer of female primordial germ cells. These results indicate that primordial germ cells isolated from embryonic blood can differentiate into functional gametes giving rise to viable offspring in the gonads of opposite-sex recipient embryos and chickens, although the efficiency was very low.

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A 95-base-pair immedte upstream sequence of the human class II major histocompatibility complex DQB gene containing the conserved X and Y elements showed enhancer activity in a transient expression assay. An "enhancer test plsmid" harboring the bacterial chloramphenicol acetyltransferase gene under the control of a truncated simian virus 40 enhancerless early promoter was employed. The DQB sequence inserted into this plasmid was active as an enhancer (9) can also affect the expression levels of these genes. Coordinate expression of the class II genes suggests a common regulatory mechanism(s). Mutant B-cell lines that lack the expression of all class II genes have been isolated by somatic cell mutagenesis (10-12). Cell fusion experiments using these cells have shown that the class II gene expression of these cells can be rescued by trans-acting factor(s) provided by class II-positive mouse B cells (13) and T cells (14). A complementation study using several such mutant cell lines, including one from a patient with class Il-negative bare-lymphocyte syndrome, has suggested that there are at least two distinct trans-acting factors specifically required for the expression of the class II genes (15).Two highly conserved sequences, termed X and Y boxes, occur in the immediate upstream regions of all of class II genes of humans and mice, and their role in the regulation of class II gene expression has been suggested (16, 17). In the DQB gene used in this study, the conserved box X is located from -113 to -100 base pairs (bp) relative to the cap site and the conserved box Y is located from -81 to -67 bp (18) (see Fig. lA). A study by gel retardation and DNase I "footprinting" assays has identified several protein factors, some of which are B-cell-specific, in nuclear extracts from various types ofcells that bind to the X or Y box sequence of the DQB gene (19). Several other studies using the DRA gene (20) 6909The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

No abstract

The octopine tumor-inducing (Ti) plasmid pTiA66 has an insertion mutation in its T region (the DNA region incorporated into the plant genome) that results in the slow growth of crown gall tumors. These tumors exhibit hormonal autonomy different from that of the crown gall tumors caused by wild-type Ti plasmids. In the present study, the nucleotide sequences of both the DNA segment inserted into pTiA66 and its target site have been determined. The inserted segment is 2548 base pairs long and has 20-base-pair terminal inverted repeats. An 8-base-pair sequence at the target site is duplicated at both integration junctions. These structural features of the insert suggest that it is a bacterial insertion sequence (IS) element, which we have named IS66. Blot-hybridization analyses using IS66 probes revealed that genomes of octopine Ti plasmids contain at least three sequences homologous to IS66: two homologues are located in the virulence region and one is located between the left-hand (TL-DNA) and right-hand (TR-DNA) portions of T-DNA. The chromosome of Agrobacterium tumefaciens A66 also contains two sequences highly homologous to IS66. These results suggest that the mutant pTiA66 plasmid was generated by translocation of one of the sequences showing homology with IS66 into the T region. The fact that a sequence homologous to IS66 is present between TL-DNA and TR-DNA also suggests that the octopine T region was split into two portions, TL-DNA and TR-DNA, by translocation of IS66 or its relatives. Thus, IS66 may cause genetic and structural variations of the T region and the vir region of the octopine Ti plasmids.Tumor-inducing (Ti) plasmids harbored by oncogenic strains of Agrobacterium tumefaciens cause formation of tumors on dicotyledonous plants, called crown galls (1). Cells isolated from the crown gall tumors are characterized by their unlimited proliferation in medium lacking phytohormones, such as cytokinins and auxins, that are required for the culture of normal plant cells (2). It has been shown that a specific DNA region of the Ti plasmid, called the T-DNA region (Fig. 1), causes tumor formation when transferred into plant chromosomes and that the T-DNA persists in the chromosomes of the tumor cells (3-6). The virulence (vir) region of the Ti plasmid (Fig. 1) is located outside the T region and is also required for tumorigenesis by the Ti plasmid. However, this region is probably not stably maintained in the tumor cells (7-10).The Ti plasmids are classified on the basis of the novel amino acid metabolites, such as octopine or nopaline, whose syntheses are directed by T-DNA genes in the tumor cells (11-15). The octopine T region is composed of twQ DNA segments near each other in the Ti plasmid (16) (Fig. 1), whereas the nopaline T region consists of one contiguous DNA segment (17, 18). The chromosomes of the tumor cells caused by the octopine strain contain either one or two portions of the T region (16). All tumor cells that have been analyzed contained the left portion of T-DNA (TL-DNA) but not all...

Petitte, J.N., Clarck, M.E., Verrinder Gibbins, A. M. and R. J. Etches (1990; Development 108, 185–189) demonstrated that chicken early blastoderm contains cells able to contribute to both somatic and germinal tissue when injected into a recipient embryo. However, these cells were neither identified nor maintained in vitro. Here, we show that chicken early blastoderm contains cells characterised as putative avian embryonic stem (ES) cells that can be maintained in vitro for long-term culture. These cells exhibit features similar to those of murine ES cells such as typical morphology, strong reactivity toward specific antibodies, cytokine-dependent extended proliferation and high telomerase activity. These cells also present high capacities to differentiate in vitro into various cell types including cells from ectodermic, mesodermic and endodermic lineages. Production of chimeras after injection of the cultivated cells reinforced the view that our culture system maintains in vitro some avian putative ES cells.

No abstract

The plasmid DNA, pAcZ, containing Escherichia coli beta-galactosidase (lacZ) gene under the control of chicken beta-actin gene promoter was injected in a linearized form into the germinal disc of fertilized chick ova at the single-cell stage. The manipulated embryos were cultured by the method of Naito et al. (1990) until hatching. The rate of hatching was 11.8% (31/263), and 19 males and 6 females were matured. DNA from blood and semen samples of the 25 matured chickens was analyzed for the presence of the injected DNA by Southern blot hybridization. The injected DNA was detected in the blood DNA of one male and in the sperm DNA of another male up to 13 months after hatching, indicating that the injected DNA was stably maintained in these chickens. Restriction digestion analysis of the injected DNA suggested that it was not rearranged and was organized as head-to-tail multimers. The copy numbers of the DNA were 0.07-0.02 in the blood DNA of one male per diploid genome, and 0.02-0.015 in the sperm DNA of another male, indicating that the exogenous DNA was present in limited populations of blood and sperm cells.