Molecular mechanisms of ventricular hypoplasia.

We have established the beginnings of a road map to understand how ventricular cells become specified, differentiate, and expand into a functional cardiac chamber (Fig. 5). The transcriptional networks described here provide clear evidence that disruption of pathways affecting ventricular growth could be the underlying etiology in a subset of children born with malformation of the right or left ventricle. As we learn details of the precise mechanisms through which the critical factors function, the challenge will lie in devising innovative methods to augment or modify the effects of gene mutations on ventricular development. Because most congenital heart disease likely occurs in a setting of heterozygous, predisposing mutations of one or more genes, modulation of activity of critical pathways in a preventive fashion may be useful in averting disease in genetically susceptible individuals.

Interaction of the nuclear matrix-associated region (MAR)-binding proteins, SATB1 and CDP/Cux, with a MAR element (L2a) in an upstream regulatory region of the mouse CD8a gene.

Matrix-associated regions (MARs), AT-rich DNA segments that have an affinity for the nuclear matrix, have been shown to play a role in transcriptional regulation of eukaryotic genes. The present study demonstrates that a DNA element, called L2a, which has been implicated in the transcriptional regulation of the mouse CD8a gene encoding an important T cell coreceptor, is a MAR. Moreover, the identities of two nuclear proteins, L2a-P1 and L2a-P2, previously shown to bind to the L2a element, have been determined. The L2a-P1 protein found to be present in all CD8-positive T cell lines tested is SATB1, a known MAR-binding protein. The widely expressed L2a-P2 protein is CDP/Cux, a MAR-binding protein that has been associated with repression of gene transcription. Interaction of both proteins with the L2a element was studied using the missing nucleoside approach, DNase I footprinting, and electrophoretic mobility shift assays with wild type and mutant L2a elements. The data suggest that CDP/Cux bound to the L2a element is displaced by binding of SATB1 and the accompanying conformational change in the DNA lying between the primary binding sites of SATB1 and CDP/Cux. We suggest that displacement of CDP/Cux by SATB1 favors transcription of the CD8a gene, possibly by enhancing or altering its association with the nuclear matrix.

The Bop gene adjacent to the mouse CD8b gene encodes distinct zinc-finger proteins expressed in CTLs and in muscle.

The Bop gene (for CD8b opposite) is located immediately upstream of the mouse CD8b gene. Expression of Bop gene transcripts was previously observed in several long term CTL lines and in thymus. The present studies demonstrate that expression of the Bop gene in lymphocytes appears to be confined to CD8-positive cells, and that Bop gene expression is inducible by Con A. They further show that a single Bop gene encodes protein products with distinct amino-terminal sequences that are expressed in CTLs (t-BOP) and in cardiac and skeletal muscle (skm-BOP), as well as what appears to be a noncoding cDNA (t-ncb) expressed only in CTLs. The t-BOP and t-ncb cDNAs in CTLs result from alternative splicing of a single primary transcript, whereas the Bop transcripts expressed in CTLs and in muscle appear to be transcribed from different promoters. The BOP proteins expressed in CTLs and muscle contain zinc finger-like motifs with homology to those of the ETO/MTG8 proto-oncogene and several other proteins of interest. Western blot analysis with a hamster anti-BOP mAb have detected the BOP protein in muscle cells and in COS 7 cells transfected with Bop cDNA constructs.

Bop: a new T-cell-restricted gene located upstream of and opposite to mouse CD8b.

In the course of transient expression studies undertaken to determine the location of the mouse CD8b gene promoter, two additional promoter activities were detected within 600 nucleotides upstream of the gene. One activity directs transcription in the same direction as CD8b but fails to transcribe the CAT reporter gene due to an apparent transcription-blocking element lying between it and the gene. The second activity directs transcription opposite to that of the CD8b gene. Northern hybridization with a probe consisting of nucleotides -875 to -550 relative to the site of CD8b transcription initiation revealed hybridizing species of 4 kilobases (kb) and 1.8 kb in poly-A-selected RNA from mouse thymus but not from any other tissues. Similar RNA species were detected in poly-A+ RNA from concanavalin A-stimulated spleen cells and several long-term CTL lines but not from the EL4 or BW5147 T-cell lines or the J558L myeloma. The mRNA species were most abundant in cells of a secondary mixed leukocyte culture which were greater than 95% CD8(+). Northern hybridizations using single-stranded unidirectional probes indicated that these mRNAs represent transcription opposite to the CD8b gene. The tissue and cell type distribution of this newly-discovered gene (designated Bop for CD8b opposite) are consistent with T-cell-specific and possibly CD8-positive T-cell-specific expression. The head-to-head arrangement of the Bop and CD8b genes is reminiscent of the arrangement of the Tap1 and Lmp2 genes, and the expression of the Bop gene in CD8-positive cells raises the possibility that these genes are involved in the same functional pathway.

Delivery of exogenous antigen into the major histocompatibility complex class I and class II pathways by electroporation.

Exogenous, nonreplicating protein antigens (Ags) are usually taken up by antigen-presenting cells (APCs) via endocytosis or pinocytosis and enter the major histocompatibility complex (MHC) class II processing and presentation pathway. Although exogenous Ags are not processed and presented in the class I pathway by most cells, soluble proteins can enter the class I processing and presentation pathway if they are introduced directly into the cytoplasm of APCs. The purpose of these studies was to determine whether exogenous proteins could be processed and presented to T cells if they were delivered into cells by electroporation. The conditions for electroporation were optimized so that the viability of the electroporated cells was high, and the majority of electroporated cells had protein incorporated. Electroporated B cells not only presented exogenous ovalbumin to CD8+, class I MHC-restricted T cells but also stimulated CD4+, class II MHC-restricted T cells. Electroporated cells also primed Ag-specific cytotoxic T lymphocytes (CTLs) in vivo, stimulated CTL precursors in vitro, and served as target cells for lysis by Ag-specific CTLs, indistinguishable from transfected cells. Thus, electropermeabilized cells were structurally intact, and the introduced exogenous protein was processed and presented in association with both class I and class II MHC molecules. This approach is as efficient and reproducible as other techniques of delivering exogenous proteins into the intracellular processing pathways. These studies suggest that electroporation could be employed for the study of cell-mediated immunity to various exogenous proteins.

Induction by concanavalin A of specific mRNAs and cytolytic function in a CD8-positive T cell hybridoma.

A previous report from this laboratory described the production of CD8+, class I-specific T cell hybridomas which developed specific cytolytic activity and the ability to secrete IL-2 upon Con A or specific Ag stimulation. Unlike normal lymphocytes or long-term CTL lines for which exposure to Ag triggers both differentiation and proliferation, T cell hybridoma lines can be activated functionally against a background of continuous proliferation. They therefore provide a unique system with which to study the molecular events involved in the induction of cytolytic function. The expression of mRNA from a series of genes was evaluated by Northern hybridization at various times after Con A stimulation of the H-2Ld-specific CD8+ 3D9 hybridoma. Induction of the c-fos proto-oncogene by 45 min poststimulation was followed shortly by c-myc induction. Perforin mRNA was expressed at a low level in the unstimulated hybridomas, but was down-regulated upon Con A stimulation to levels undetectable by PCR. Interestingly, production of granzyme A mRNA was strongly induced by 45 min after Con A stimulation. In the CD8+ RT-1.3G3 hybridoma, which is nonlytic and specific for the HIV-1 envelope glycoprotein, c-fos but not granzyme A mRNA was induced by 45 min poststimulation, and no granzyme A mRNA was detectable at any time. Thus, a significant role for granzyme A in the induction of cytolytic activity is suggested. Cytolysis by the 3D9 hybridoma involved both target cell membrane damage and DNA fragmentation, and both Ca(2+)-dependent and Ca(2+)-independent cytolysis were observed. Although TNF-alpha mRNA was induced by 4 h poststimulation, Ab to TNF-alpha failed to inhibit the Ca(2+)-independent lysis observed, leaving the basis for the observed Ca(2+)-independent lysis unexplained.

Cis-acting DNA elements and cell type-specific nuclear proteins which may play a role in regulation of mouse CD8 alpha (Lyt-2) gene transcription.

Fusion of mouse CD8+ class I MHC-restricted T cells with the BW5147 thymoma invariably yields CD8- hybridomas in which RNA transcribed from the CD8 alpha (Lyt-2) gene is undetectable. To determine whether cis-acting DNA sequences may negatively regulate transcription of the Lyt-2 gene in BW5147 cells, one possible explanation for the above observation, BW5147 cells were stably transfected with the Lyt-2 gene containing 1 - 11,000 nucleotides of 5' flanking DNA and surface expression of Lyt-2 was monitored by flow microfluorometry. Initial results suggested the presence of a negative element between 1400 and 5000 nucleotides upstream of the site of transcription initiation. Further studies suggested the presence of two potential negative regulatory elements in this region, one of which includes a 269 nucleotide Accl - SstI fragment comprised of nucleotides -4700 to -4431 which bound nuclear proteins from CD8+ and CD8- cell lines in electrophoretic mobility shift assays (EMSA). EMSA studies performed using nuclear extracts from a variety of cell lines and tissues demonstrated that unique retarded complexes, called bands 1 and 2, correlated significantly with expression or non-expression of Lyt-2 respectively. EMSA analysis of proteins fractionated by SDS-PAGE from nuclear extract of the CD8+ VL3 T lymphoma cell line revealed proteins of approximately 110-130 kDa (called L2a-P1) and > 200 kDa (called L2a-P2) which bind within a 100 nucleotide region of this fragment (called L2a) to yield band 1 and 2 respectively, and which may play a role in regulation of Lyt-2 gene transcription.

Identification of a rel-related protein in the nucleus during the S phase of the cell cycle.

The c-rel proto-oncogene encodes a 75-kDa protein (p75c-rel) which is present in the cytosol of chick embryo fibroblasts (CEF) associated with a distinct set of cellular proteins with molecular masses of 40, 115, and 124 kDa. CEF cultures arrested in S phase of the cell cycle, or enriched for G2 or mitotic cells, were examined to determine whether the expression of c-rel was altered during the cell cycle. Levels of p75c-rel remained constant in all portions of the cell cycle examined; however, a Rel-related protein with an apparent molecular mass of 64 kDa was detected in nuclei of S-phase cells. As cells enter G2, the level of this protein in the nucleus decreases. This protein reacts with antiserum generated against the carboxy terminus of p75c-rel in radioimmunoprecipitations and Western immunoblot experiments and was also detected in a Western immunoblot with antiserum generated against the first 161 amino acids of pp59v-rel. Thus, unlike other Rel/NF-kappa B family members, p64 has carboxy-terminal homology with c-Rel. The majority of peptides generated by partial proteolytic cleavage of p64 are shared with peptides generated by digestion of p75c-rel and/or pp59v-rel. We suggest that this protein represents a new member of the Rel family of transcription factors and is located in the nucleus of avian fibroblasts during S phase of the cell cycle.

A dominant epitope of HIV-1 protease recognized by hamster monoclonal antibodies.

Monoclonal antibodies (mAbs) against HIV-1 protease (HIV PR), the essential protease of human immunodeficiency virus type 1 (HIV-1), were produced in Armenian hamsters. Studies of direct binding to synthetic peptides and inhibition of binding to intact protease by peptide competition showed that five mAbs recognized an epitope that includes the sequence LPGRWKPK (residues 38-45), which lies near the region of the protease called the flap. All of the mAbs react specifically with HIV PR in Western blots. Because of structural conservation of the epitope in the proteases of many HIV-1 isolates, mAbs to this epitope are likely to be useful for detection of HIV PR in field isolates of HIV-1. Also, mAbs specific for this epitope, which lies close to the flap of HIV PR, may be useful for functional studies of HIV PR and possibly for the design of inhibitors of protease activity that bind outside the enzyme's catalytic site.

Differential susceptibility of mouse Lyt-2 and Lyt-3 genes to negative regulation.

Hybrids of Lyt-2/Lyt-3-positive class I-specific cytotoxic T lymphocytes (CTLs) with the BW5147 thymoma cell line (Lyt-2/Lyt-3-negative) are known to be Lyt-2/Lyt-3-negative due to shutoff of transcription of the CTL's Lyt-2 gene. Hybrids of a constitutively Lyt-2-positive transfectant of BW5147 (3B2) with a long term CTL line, 2C, and with CTLs generated in a mixed leucocyte reaction (MLR) shut off the CTL's Lyt-2 gene as expected but express the CTL's Lyt-3 gene product as a heterodimer with the product of the transfected Lyt-2 gene. Thus the Lyt-3 gene is not subject to the same negative regulatory influences as the Lyt-2 gene. That expression of Lyt-2 is not necessary for Lyt-3 gene transcription to continue is demonstrated by the finding that hybrids of MLR-generated CTLs with either BW5147 (Lyt-2-negative) or 3B2 (Lyt-2-positive) cells express Lyt-3 RNA. Southern hybridization and structural analysis of DNA fragments generated using the polymerase chain reaction demonstrated that hybrids contain several species of Lyt-3 RNA, one of which lacks the exon encoding the extracellular V-like domain and appears to be the product of an alternatively-spliced RNA transcript.

A self-reactive T cell population that is not subject to negative selection.

In male mice expressing a transgenic alpha beta TCR which recognizes a male antigen (HY), T cells which do not express normal levels of CD8 escape thymic deletion and appear in the periphery. These consist of two distinct populations, one which lacks expression of both CD4 and CD8, and one with low levels of CD8. Neither population has anti-HY reactivity, consistent with the known requirement of this TCR for CD8. We now describe the consequences of expression of both the anti-HY TCR transgene and a constitutive CD8.1 transgene on T cells of male mice. Peripheral T cells in these male 'double transgenic' mice express both the anti-HY TCR and normal levels of CD8, and can proliferate to male antigen in vitro. These cells do not express the endogenous allele of CD8 (CD8.2), suggesting that the increase in CD8 levels due to the CD8.1 transgene leads to the deletion of the CD8.2low population. In contrast, the CD8.1 transgene does not lead to the deletion of the CD8.2- population. This implies that, unlike the majority of alpha beta T cells, TCR+CD4-CD8- cells in TCR transgenic mice are not subject to deletion.

Inducible functions in hybrids of a Lyt-2+ BW5147 transfectant and the 2C CTL line.

Cytolytic activity and release of interleukin 2 (IL-2) were induced in Lyt-2-positive T-T cell hybrids by incubation with either concanavalin A or irradiated stimulator cells. Since hybrids of Lyt-2-positive class I-specific cytotoxic T lymphocytes (CTLs) with the fusable mouse thymoma cell line, BW5147, are invariably Lyt-2-negative, a derivative of BW5147 was produced by transfection which constitutively expresses surface Lyt-2.1. This cell line, 3B2, was fused with the H-2Ld-specific long term CTL line, 2C. Such hybrids expressed the transfected Lyt-2 gene but not the endogenous gene of the 2C fusion partner. That Lyt-2 plays a functional role in hybrids of 3B2 with 2C is shown by the observations that: 1) cytolysis by Lyt-2-positive hybrids was inhibited by Lyt-2-specific monoclonal antibody (mAb); 2) Lyt-2-positive but not Lyt-2-negative subclones of one such line develop specific cytotoxicity when incubated with stimulator cells; 3) Less IL-2 was released from Lyt-2-negative subclones incubated with stimulator cells than from Lyt-2-positive subclones; 4) Lyt-2-specific mAb inhibits release of IL-2 from Lyt-2-positive hybrids incubated with stimulator cells. All Lyt-2-positive hybrids expressed functional surface Lyt-3 encoded by the CTL fusion partner, demonstrating that expression of the Lyt-3 gene is not sensitive to the negative regulation which shuts off the endogenous Lyt-2 gene in hybrids of class I-specific CTLs with the 3B2 or BW5147 cell lines. The existence of inducible T-T cell hybrids expressing functional Lyt-2 and Lyt-3 provides a system for evaluation of the role(s) of Lyt-2 and Lyt-3 in the induction of function independent of cell growth.

Polymorphism in V kappa 10 genes encoding L chains of antibodies bearing the Ars-A and A48 cross-reactive idiotypes.

p-azophenylarsonate-specific antibodies of A/J mice which bear the Ars-A crossreactive idiotype utilize the V kappa-Ars-A gene segment, a member of the V kappa 10 family. Southern hybridization of genomic DNA from several inbred strains using a probe from the 5' flanking region of the V kappa-Ars-A gene demonstrated three patterns of restriction fragment length polymorphisms (RFLP). Six genes corresponding to hybridizing bands were obtained from DNA libraries of C.AKR, PERU and A/J mice, and nucleotide sequence comparisons revealed two allelic groups: AKR1 (Igk-V10.1a), AJ1 (Igk-V10.1b) and PERU1 (Igk-V10.1c); AKR2 (Igk-V10.2a), AJ2 (Igk-V10.2b), and PERU2 (Igk-V10.2c). The Igk-V10.1b gene of the A/J strain is the V kappa-Ars-A gene used in Ars-A idiotype-positive antibodies. The product of the C.AKR allele (Igk-V10.1a) contained four amino acid substitutions in CDR3 as compared with Igk-V10.1b. These substitutions probably explain the failure of AKR mice and other strains with the same V kappa 10 RFLP pattern to provide in genetic crosses a L chain which, together with the A/J VH-ArsA gene product, form Ars-A idiotype-positive antibodies. Also, the nucleotide sequence identity between the Igk-V10.1c and Igk-V10.1b alleles and the Igk-V10.2c and Igk-V10.2b alleles is significantly greater than that seen in comparisons with the Igk-V10.1a and Igk-V10.2a alleles, respectively, suggesting an evolutionary pathway similar to that of the linked Igk-J locus. BALB/c antibodies bearing the A48 regulatory idiotype contain L chains encoded by the BALB/c Igk-V10.1b and Igk-V10.2b alleles. Strongly A48 idiotype-positive antibodies utilize the Igk-V10.1b chain, and weakly A48-positive antibodies use the Igk-V10.2b L chain. The possible effects of amino acid substitutions specified by the Igk-V10.1a, Igk-V10.1c, Igk-V10.2a, and Igk-V10.2c alleles on their ability to provide L chains used in A48 idiotype-positive antibodies are discussed.

Lymphomas with acquired mouse mammary tumor virus proviruses resemble distinct prethymic and intrathymic phenotypes defined in vivo.

A number of murine T cell lymphomas expressing the T cell Ag Thy-1 contain acquired mouse mammary tumor (MMTV) proviruses. These lymphomas all express detectable levels of MMTV RNA, yet the majority of the tumors fail to produce MMTV particles. To determine if the ability of lymphomas to produce MMTV is a reflection of the differentiation state of the tumor, we examined eight lymphomas for expression of surface B and T cell Ag as well as for rearrangements and expression of TCR genes. All tumors could be grouped into three categories observed in vivo, including early lymphoid, nonmature intrathymic T cells, and immature intrathymic T cells. Cell lines corresponding to all three phenotypes produced MMTV particles, suggesting that production of virus is not linked to the differentiation state of lymphoid cells. These studies highlight the potential advantage of studying T cell lymphomas vs mixed primary populations or T cell hybridomas for evaluation of both phenotypic and molecular markers in clonal T cells.

Structural and evolutionary comparisons of four alleles of the mouse Igk-J locus which encodes immunoglobulin kappa light chain joining (J kappa) segments.

The Igk-J locus of the mouse encodes the immunoglobulin kappa light chain joining (J) segments. Four Igk-J alleles have been described on the basis of restriction enzyme length polymorphisms. The nucleotide sequences of the Igk-Ja allele (type strain, C.C58), Igk-Jc allele (type strain, SJL/J), and Igk-Jd allele (type strain, SK/CamRk) have been determined and are compared with the previously reported Igk-Jb allele sequence (type strain, BALB/c). The mouse sequences are also compared with published sequences for rat and human J kappa sequences. Far more differences were found between the Igk-Ja allele and the other mouse alleles than between any two of the latter. These result in two amino acid substitutions which distinguish the J2 and J3' segments of the Igk-Ja allele from the other three alleles. Use of the Phylogenetic Analysis Using Parsimony program to generate a phylogenetic tree strongly indicates that after divergence from the rat ancestor, there appears to have been an early split between the Igk-Ja allele and the evolutionary precursor of the other mouse alleles. There also appears to have been far less divergence from the ancestral condition in the Igk-Ja allele than in the other alleles. Also, the presence of only one convergent mutation among the four mouse alleles provides strong evidence against any crossing over within the Igk-J locus during the history of these alleles. Finally, the differences in rates of evolution of the Igk-J alleles are in marked contrast to the relatively uniform rates of divergence of four alleles of a mouse V kappa gene, Igk-VSer.

Structural and evolutionary comparisons of four alleles of the mouse immunoglobulin kappa chain gene, Igk-VSer.

The mouse Igk-VSer gene encodes an immunoglobulin kappa light chain variable region which gives rise to two phenotypic polymorphisms of mouse kappa chains. The nucleotide sequences of coding and flanking regions of the Igk-VSerc and Igk-VSerd alleles found in recently inbred strains of wild mice are compared with those of the Igk-VSera and Igk-VSerb alleles described previously. Results suggest that the gene is evolving randomly and that framework 2 and complementarity determining region 2 are preserved, presumably for overall light chain structure. Results indicate that all four alleles have an octamer motif upstream of the gene which should be functional and allow prediction of whether or not the product of the germ line gene will be detectable as either the IB-peptide or Ef1a phenotypic polymorphism. Southern hybridization of genomic DNA using as probe a 1-kb Xba I-Xba I fragment located approximately 4 kb upstream of the BALB/c Igk-VSerb coding region demonstrated the presence of homologous DNA in mice bearing the Igk-VSera allele and absence from mice bearing the Igk-VSerc and Igk-VSerd alleles. Nucleotide sequence comparison of BALB/c and SK/CamRk (Igk-VSerd) DNA in this region demonstrated that BALB/c contained an insertion 2.4 kb in length which was absent from SK/CamRk. Both strains contain DNA homologous to the reverse complement of the mouse Bam5 repetitive element at the point of the insertion, with BALB/c containing approximately 70 nucleotides more of the element than SK/CamRk. Surprisingly, the strains containing DNA related to the Xba I-Xba I probe are not those determined to be the most similar by nucleotide sequence comparisons and by the Phylogenetic Analysis Using Parsimony program. The evolutionary relationship of the alleles and a possible basis for the inconsistency presented by the Xba I-Xba I fragment-related DNA are discussed.

Nucleotide sequence analysis of the C.AKR Lyt-2a gene: structural polymorphism in alleles encoding the Lyt-2.1 T-cell surface alloantigen.

The Lyt-2a allele of the C.AKR strain of mice (genotype Lyt-2a, Lyt-3a) was cloned, and its complete nucleotide sequence as well as that of 2 kb of 5' flanking DNA was determined. The sequence was compared with the partial sequence of the Lyt-2a allele of DBA/2 (genotype Lyt-2a, Lyt-3b) and the nearly complete sequence of the B10.CAS2 Lyt-2b allele reported by Liaw and coworkers (1986). The coding regions of the two Lyt-2a alleles differ from each other by two nucleotide substitutions in the three exons over which they could be compared, resulting in two amino acid substitutions in the leader and transmembrane segments. The coding region of the C.AKR Lyt-2a allele differs from the Lyt-2b allele by two nucleotide substitutions in the extracellular V-like domain, one of which is silent and the second of which leads to substitution of valine for methionine at amino acid position 78 giving rise to the Lyt-2.1 allotypic specificity. The coding region of the DBA/2 Lyt-2a allele shares with C.AKR the allotypic substitution at position 78 and differs from Lyt-2b by three additional nucleotide substitutions in the coding regions, two of which lead to amino acid substitutions in the leader and transmembrane segments. It would therefore appear that the Lyt-2 alleles of the three strains analyzed are distinct, and the nomenclature Lyt-2a1 and Lyt-2a2 is suggested to distinguish the alleles of C.AKR and DBA/2, respectively. These alleles share a common difference from the Lyt-2b gene product at position 78, and since the amino acid substitutions which distinguish them from each other are in the leader and transmembrane segments, their mature Lyt-2 gene products appear antigenically identical.

Analysis of the effect of a strain-specific alteration in the upstream octamer region on transcription of a mouse V kappa Ser light chain gene.

Previous studies had shown that several phenotypic markers expressed by several strains of mice (C58, AKR, PL, RF) involve a light chain group, called V kappa Ser, encoded by a single germ-line gene (Igk-VSera). Most other inbred strains (e.g., BALB/c) contain an allele (Igk-VSerb) which differs from Igk-VSera both in coding regions and in an upstream octamer region known to be important for transcription. Since no evidence for expression of the Igk-VSerb gene product has been observed, experiments were undertaken to determine whether the alteration in the regulatory octamer region of BALB/c might have rendered it defective for transcription. The upstream octamer-containing region of a cloned functional V kappa Ser gene expressed by the C.C58 myeloma M75 was replaced by the corresponding region from BALB/c or deleted entirely. Constructs were transfected into J558L cells and quantity of transcription and site of transcription initiation were compared. Results suggest that the BALB/c octamer (CTTTGCGT), which differs at two of eight nucleotides from the consensus octamer sequence (ATTTGCAT), is fully functional in transcription initiation. This is consistent with results of S1 nuclease protection experiments which indicate the presence of small amounts of correctly initiated V kappa Ser-related RNA in BALB/c spleen.

Structure and expression of the Lyt-3a gene of C.AKR mice.

The mouse Lyt-3a gene, which encodes the Lyt-3.1 T-cell surface alloantigen of the C.AKR strain, has been cloned, and the nucleotide sequence of its exons and more than 2 kb of 5' flanking sequence have been determined. The gene extends over approximately 16 kb of DNA and consists of six exons encoding leader, leader plus V-like domain, membrane-proximal, transmembrane, and cytoplasmic domains. The only difference between the coding region of the Lyt-3a gene and the cDNA sequences reported for Lyt-3b (Nakauchi et al. 1987. Panaccio et al. 1987) is at position 77 of the mature protein where Lyt-3a encodes serine and Lyt-3b encodes arginine. This substitution must therefore be the basis for the serological distinction between the Lyt-3.1 and Lyt-3.2 alloantigens. Potential TATA and CAAT sequences, two Sp1 protein binding sites, two extended repeats of the dinucleotide, CA, a number of short inverted repeats, and an inverted segment of the mouse B1 repetitive sequence are found 5' to the Lyt-3a gene. Two consensus poly-A addition signals and a complete copy of the mouse B1 sequence are found 3' to the gene. Both B1-related regions are flanked by short direct repeats suggesting that they arose by an insertional mechanism. Cotransfection of the Lyt-3a gene together with a cloned Lyt-2a gene resulted in expression of both Lyt-2 and Lyt-3.1 on the surface of Ltk- and BW5147 cells.(ABSTRACT TRUNCATED AT 250 WORDS)