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We have injected cloned derivatives of Xenopus laevis ribosomal genes into X. laevis oocyte nuclei and examined the resulting transcription complexes in the electron microscope. From this work we conclude that the promoter lies somewhere within a region between -320 nucleotides upstream and +113 nucleotides downstream from the site of transcription initiation. This assignment agrees with inferences based on sequence conservation. It further suggests that the duplicated initiation region sequences located further out in the spacer ("Bam islands") are not required for the normal high densities ofRNA polymerase loading seen on ribosomal genes. Concerning termination, the cluster of four Ts that forms part of the HinduI restriction site at the 3' end of the gene appears to be part of the normal termination signal. Termination still occurs when ofily three Ts are present, but reduction to two Ts damages termination. Because clusters of three Ts appear at several sites within the gene, it is likely that sequences adjacent to the T cluster also are required for normal termination. In addition, we present evidence for a fail-safe termination site just upstream from the site of transcription initiation.The genes coding for the large ribosomal RNAs (the rDNAs) from the frog Xenopus laevis have been the subject ofintensive study during the last decade. This work has culminated recently in determination ofthe nucleotide sequence oflarge parts ofthe repeating unit (1-4), identification ofthe primary transcript (5), and localization ofthe precise sites ofinitiation and termination of transcription (2). In this paper we begin the mapping of the nucleotides involved in promoter and terminator function in the ribosomal genes.Mapping of promoter and terminator-sequences requires a transcription system in which to measure the activity of genes with suitable deletions or other mutations. The approach that we have used is to inject cloned ribosomal genes into oocyte nuclei and to assay transcription ofthese genes by spreading the nuclear contents for electron microscopy, using the methods of Miller and Bakken (6). Such direct visualization of transcribing ribosomal gene plasmids, as reported by Trendelenburg and Gurdon (7), allows one to observe certain features of transcriptional'behavior that would'be difficult to study by biochemical methods.In this paper we compare the specificity and frequency of initiation on a plasmid that bears a complete ribosomal gene and spacer, repeat with that on a ribosomal gene from which much of the DNA surrounding the initiation site has. been deleted. We also examine the efficiency of termination in recombinant plasmids with altered sequences at the 3' end of the ribosomal gene. MATERIALS AND METHODSOocyte Injection. Stage V-VI X. laevis oocytes (8) were used for injections. Biochemical measurements have shown that these stages are still highly active in ribosomal RNA synthesis (9, 10). We have noticed, however, that the structure of the ribosomal genes can be quite variable from female to...

Stlmma.ryA major obstacle to the development of T cell therapy for the treatment of human tumors has been the difficulty generating T cells specifically reactive with the tumor. Most of the characterized human tumor antigens have been dassified as tumor associated, because of demonstrable expression at low levels in some normal cells, and thus have not been extensively studied as potential targets of a therapeutic immune response. However, the quantitative difference in expression of such antigens between the tumor and normal cells might permit the generation of antigen-specific T cells capable of selective antitfimor and not autoimmune activity. To address this issue, transgenic (TG) mice were generated that expressed low levels of Friend murine leukemia virus (FMuLV) envelope protein in lymphoid cells under the control of an immunoglobulin promoter. This protein is expressed at high levels by a Friend virus-induced erythroleukemia of C57BL/6 (B6) origin, FBL, and has been shown to serve as an efficient tumor-specific rejection antigen in B6 mice. The env-TG mice were tolerant to envelope, as reflected by the failure to detect an envelopespecific response after in vivo priming and in vitro stimulation with preparations of FMuLV envelope. However, adoptively transferred envelope-specific T cells from immunized non-TG B6 mice mediated complete eradication of FBL tumor cells in TG mice, and did not induce detectable autoimmune damage to TG lymphoid tissues. The transferred immune cells were not permanently inactivated in the TG mice, since donor T cells responded to envelope after removal from the TG mice. The lack of autoimmune injury did not reflect inadequate expression of envelope by TG lymphocytes for recognition by T cells, since TG lymphocytes functioned effectively in vitro as stimulators for envelope-specific T cells. The results suggest that this and analogous strains of TG mice may prove useful for elucidating principles for the generation and therapeutic use of tumor-reactive T cells specific for tumor-associated antigens.

Factor IX is the precursor of a serine protease that functions in the intrinsic blood clotting pathway. Deficiencies in this plasma glycoprotein result in haemophilia B (or Christmas disease) and occur in about 1 in 30,000 males. Patients are currently treated with fresh frozen plasma or prothrombin complex concentrates prepared from pooled plasma from normal individuals. There are several problems with this method of treatment, including the probable exposure of the patients to contaminants such as the viral agents responsible for hepatitis and AIDS (acquired immune deficiency syndrome). As a first step towards an alternative source of pure human factor IX, we report here on the use of recombinant DNA techniques to produce biologically active factor IX in cultured mammalian cells. Stable cell lines were produced by cotransfecting a baby hamster kidney (BHK) cell line with a plasmid containing a gene for factor IX and a plasmid containing a selectable marker. Protein secreted by these cell lines reduces the clotting time of plasma from factor IX-deficient patients. We present additional evidence that this protein is authentic human factor IX.

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