Fung-Win Shen

A pseudogene homologous to mouse transplantation antigens: Transplantation antigens are encoded by eight exons that correlate with protein domains

We have isolated about 30 to 40 different BALB/c mouse sperm DNA genomic clones that hybridize to cDNA clones encoding proteins homologous to transplantation antigens. One of these clones (27.1) was selected for sequence because it was polymorphic in Southern blot analysis of the DNAs from BALB/c and CBA mice. A fragment of 5.7 kilobases of this clone was completely sequenced and found to contain a pseudogene whose sequence is highly homologous to the sequences of known transplantation antigens. Pseudogene 27.1 is split into eight exons that correlate with the structurally defined protein domains of transplantation antigens. Using Southern blot hybridization on the DNAs of different inbred mouse strains, we mapped the pseudogene to the Qa-2,3 region, a part of the Tla complex on chromosome 17 that is adjacent to the major histocompatibility complex. The Qa2,3 region encodes lymphoid differentiation antigens homologous to the transplantation antigens in size, in peptide map profiles and in their association with beta2-microglobulin. These mapping studies suggest that gene 27.1 may be a pseudogene for eigher a Qa antigen or an as yet undefined transplantation antigen. Accordingly, we may have isolate genes encoding lymphoid differentiation antigens of the Tla complex as well as those encoding transplantation antigens among the 30 to 40 different genomic clones isolated from our sperm library.

Genetic nomenclature for loci controlling mouse lymphocyte antigens

There are now over fifty serologically defined mouse lymphocyte cell-surface antigens. Of these, more than thirty are polymorphic and have been shown to be controlled by a single genetic locus. These loci have mostly been designated by the symbol Lyfollowed by a number assigned more or less in order of discovery of the locus (Table 1). However, there has been no mechanism for orderly assignment of symbols or numbers, with consequent use of a variety of symbols, duplication of numbers, and use of different symbols or numbers for the same locus. The authors recently met at the National Institutes of Health, Bethesda, Maryland, to discuss means of overcoming these problems. A preliminary draft of nomenclature hales for the Ly loci was prepared and circulated among colleagues. In this paper we present a revised version of the rules in which we (1) reallocate duplicated Ly numbers, (2) provide a definition of Ly loci, and (3) establish a central Ly nomenclature registry that will be responsible for coordinating the allocation of new Ly numbers. In establishing this nomenclature, we have tried to maintain the status quo and have only reallocated duplicated numbers or renamed loci with grossly misleading names in order to cause minimum alteration of the existing system. Thus, for example, Lna, Tind, Tsu, Thb, and Pca-1, which are informative names, have not been altered.

A gene in the H-2S:H-2D interval of the major histocompatibility complex which is transcribed in B cells and macrophages.

In screening a mouse B-cell cDNA library for clones representing genes selectively expressed by B cells, we encountered a clone, B144, which appears to refer to a previously unrecognized gene between H-2S and H-2D. Some properties of this gene, which is not obviously classifiable with other varieties of genes in the major histocompatibility complex (MHC), are described in this report. The B-cell cDNA library, containing .-~ 1.4 × 10 6 independent transformants, was made using the pCD vector (Okayama and Berg 1983) and poly(A) + mRNA of 1.29 cells. 1.29 is a B-cell spontaneous ascites tumor that originated in an I/St/Boy mouse. About 1 x 105 colonies of a sublibrary with insert sizes of 0.5-1.5 kb were screened with 32p-labeled 1.29 cDNA previously subtracted with mRNA of T-cell leukemias ISL57 and ASL1, sarcoma Meth A, and BALB/c liver. Positive clones were hybridized first with combined cDNA of B cells (NFS-1.3 and NFS-5) and then with combined cDNA of ISL57, ASL1, and Meth A. Colonies hybridizing preferentially with B-cell cDNA were selected. To eliminate clones representing known class II genes, colonies were further hybridized with a combination of 32p-labeled class II clones [pAAC6 (A~) (Benoist et al. 1983), A¢1/SP64-1 and A~2/Sp64-2 (Wake and Flavell 1985), cEBs2 (Ee) (Mengle-Gaw and McDevitt 1985) and 32.1 (E~) (3.4 kb Sal I fragment) (Steinmetz et al. 1982)]. This yielded 26 cDNA clones from which plasmid DNAs were isolated and used as probes in Northern blotting to assess selective expression by B cells. Three clones appeared specific for B cells but were found to represent an IgX2 gene and were not studied further. However, a further clone, B144, appeared to represent a novel or unidentified gene that is transcribed in B cells and macrophages. In Northern blotting with a nick-translated B144 probe, a single band of ~ 800 nucleotides was observed with total RNA of spleen and lymph node cells and of B and macrophage cell lines, but not o fT and other cell types named in Figure 1 nor of T leukemias ASL1, EL4, and K36 (not shown). In case a known class II gene had escaped screening, Northern blots representing B144, A~, and E~ were compared. The B144 transcript appeared shorter than the 1.3 kb of usual class II transcripts and shorter than the reported lengths for CR3 complement receptor (Sastre et al. 1986) and FcT-receptor (Lewis et al. 1986) which also are selectively transcribed in B cells and/or macrophages.

Ly-1 inducer and Ly-1,2 acceptor t cells in the feedback suppression circuit bear an I-J-subregion controlled determinant

An I-J-subregion controlled determinant is expressed on Ly-1 inducer and Ly-1,2 acceptor T cells in the feedback suppression circuit. Ly-1 T cells absorb the I-J antibody reactive with the Ly-1,2 acceptor T cell, suggesting that both inducer and acceptor T cells have the same 1-J determinant. Since less than 10 percent of Ly-1 or Ly-1,2 T cells are killed by anti-I-J plus complement treatment, the I-J determinant demarcates functionally distinct subsets of both the Ly-1 and Ly-1,2 T-cell sets. This I-J determinant is not expressed on a detectable number of Ly-1 helper T cells which induce B lymphocytes to produce anti-sheep red cell antibody in tissue culture.

Multiple alleles of theLyb-2 Locus

The Lyb-2 antigen system was described by Sato and Boyse (1976), and the Lyb-2 locus has been mapped on chromosome 4 (Sato et al. 1977, Taylor and Shen 1977). The Lyb-2.1 alloantigen has been found only on cells of the B lymphocyte lineage. We report here two further alleles of Lyb-2. Typing for the three Lyb-2 alleles was carried out by the cytotoxicity assay on spleen cells, as described by Sato and Boyse (1976) for Lyb-2.1, with the antisera shown in Table 1. We suspected that the antisera called c~Lyb-2.2 and eLyb-2.3 in Table 1 might be recognizing Lyb-2 alleles because their reactions in the cytotoxicity assay with various suspensions were similar to those of c~Lyb-2.1 (Sato and Boyse 1976). These data are shown in Table 2. Similarly, also, absorption tests failed to reveal the presence of antigen on tissues other than those containing B cells. Tables 3 and 4 show that Lyb-2 b (Lyb-2.2) and Lyb-2 c (Lyb-2.3) segregate in F2 populations as alleles of Lyb-2 a (Lyb-2.1). Table 5 shows the strain distribution of the three Lyb-2 phenotypes. No strain has more than one Lyb-2 specificity, so there is no evidence that the Lyb-2 locus is complex. The STS/A strain expresses none of the three Lyb-2 phenotypes, so at least a fourth Lyb-2 allele remains unrecognized. Absorption tests confirm that none of the T-cell leukemias we have examined expresses Lyb-2. Neither do the three BALB/c myelomas we have t e s t e d MOPC-70A and MOPC-21 (both ~, 71) and MOPC104E (21, t~) although ccLyb-2 eliminates the majority of indirect PFC, and partially eliminates direct PFC. The Lyb-2(+) I/St spontaneous ascites tumor 1.29 (used in making Lyb-2.1 antiserum; Table 1) has cell-surface Ig but does not secrete demonstrable Ig,

Further definition of the Ly-5 system

Ly-5 is expressed by cells of the hematopoietic branch of development. Further serological analysis of the Ly-5 system, aided by Ly-5 monoclonal antibodies and by two Ly-5 congenic mouse strains, reveals two new Ly-5 alloantigens, Ly-5. 3 and Ly-5.4. The data define three thymocyte phenotypes, Ly-5.1,3, Ly-5.2,4, and Ly-5.2,3, and three corresponding genotypes, Ly-5a, Ly-5b, and Ly-5c, respectively. Ly-5ais by far the most common allele. The Ly-5callele is found only in the ST/bJ strain, a finding that accords with the presently unique pattern of restriction fragments previously observed in Southern blotting of ST/bJ DNA with an Ly-5 cDNA probe. Present serological and biochemical data favor the interpretation that the compound Ly-5 phenotype of thymocytes is attributable to two separate Ly-5 molecular isoforms that exhibit a discrete difference in protein composition, bear different Ly-5 antigens, and are produced jointly by thymocytes, unlike other Ly-5 isoforms previously shown to distinguish different hematopoietic cell lineages.

Further polymorphism of the Tla locus defined by monoclonal TL antibodies

Six new monoclonal TL antibodies are described. At least one new TL antigen is defined (TL.7), and at least one more Tla allele, bringing the total number of known Tla alleles to six. Five of the monoclonal antibodies, and probably all six, identify distinct TL antigenic specificities. Four of these antigens conform in strain distribution and expression on leukemia cells to antigens defined by conventional antisera. The data contain a hint that monoclonal TL antibodies like TL.m6 may serve to identify a region of the Tla gene, which determines whether or not prothymocytes will respond to physiological induction by expressing TL, and thus may provide a means to study the regulatory mechanism that determines whether mouse strains are phenotypically TL+ or TL−

Organization of the Ly-5 gene.

A single Ly-5 gene is known to generate a variety of transmembrane glycoprotein isoforms that distinguish various cell lineages and stages of differentiation within the hematopoietic developmental compartment of the mouse. Systems homologous to Ly-5 are known in rats and in humans. The complete exon-intron organization of the Ly-5 gene is described in this report. The Ly-5 gene occupies about 120 kilobases of chromosome 1 and comprises 34 exons, of which 32 (Ex-3 to Ex-34) are protein coding. Ex-1, Ex-2, and parts of Ex-3 and Ex-34 are untranslated. In all cDNA clones examined, either Ex-1 or Ex-2 was represented, but not both, implying that Ex-1 and Ex-2 in Ly-5 mRNA may be mutually exclusive. Primer extension and S1 nuclease protection mapping were used to identify initiation (cap) sites for transcription. The finding of putative cap sites for Ex-1 and Ex-2, and of corresponding TATA-like sequences, suggests the presence of two promoters. In both Ex-1+ and Ex-2+ cDNA clones the next exon is Ex-3, which has a translation-initiating codon. The intron between Ex-3 and Ex-4 is unusually long, about 50 kilobases. Evidence is given that Ex-5, like Ex-6 and Ex-7 (studied previously), is another alternative exon that is selectively programmed, alone or together with Ex-6 or Ex-7 or both, to generate actual or potential Ly-5 isoforms by alternative splicing.

An alloantigen selective for B cells: Ly-17.1

Alloantigen Ly-17.1 is identified by the antiserum (C3Hf/Bi x B6-Tla a) anti C3H/An spleen (~-Ly-17.1). This cell-surface antigen has not been demonstrable by cytotoxicity assay; the data given here were obtained with a pool of 15 c~-Ly-17.1 serum samples (taken after the 6th to 20th inoculations), all of which were positive by PA-SRBC assay but negative by cytotoxicity assay. The PA-SRBC assay, in which SRBC conjugated to protein A form rosettes with antibody-coated cells, was performed as originally described by Koo and Goldberg (1978). Maximum percentage counts of Ly-17 + (rosetted) cells in C3H/An mice were: spleen, 39+_5; lymph nodes, 22+_3; bone marrow, 21 +_3; thymus, 2 + 2 (e-Ly-17.1 serum dilutions up to 1 : 40; 1 x 107 cells per ml; mean of results for four mice). There were no significant differences in counts among Ly-17.1 + strains (listed below). A virtue of the PA-SRBC assay for Ly-17.1 is that control counts (spleen cells of Ly-17.1 mice, listed below) are always virtually nil, thus largely obviating the need for corrections. The counts given above may suggest that B cells are Ly-17 + and T cells L y l 7 . The data in Table 1 support this conclusion because: (a) probably all Ly-17 § cells adhere to nylon, and (b) elimination of Lyt-1 § cells, comprising 90-95 percent of splenic Thy-1 § cells, enriches the splenic Ly-17 § population to about the same degree as elution from nylon. A congenic strain, B6-Ly-17.1, is in preparation from N9 backcross segregants (allele donor C3H/An), and will shortly be available on request. Ly-17 typing of backcross segregants bred to derive this congenic strain showed: (a) 1 : 1 ratios of positive and negative Ly-17.1 phenotypes signifying single-factor inheritance; (b) complete negativity of all Ly-17.1segregants signifying no demonstrable contaminant ~ntibody to any other system represented on spleen cells, and (c) that counts of Ly-17 § cells in the PA-SRBC assay are as high with Ly-17 heterozygotes

Monoclonal antibody to an alloantigenic determinant on ?2-microglobulin (?2M) of the mouse

Polymorphism of mouse fi2M (B2m locus) has been indicated by strain differences observed in SDS-PAGE (Michaelson et al. 1980, Robinson et al. 198 la) and IEF (Goding and Walker 1980), corresponding to a single amino acid substitution (Gates et al. 1981). The B2m locus has been mapped in the region of H-3 on chromosome 2 (Michaelson 1981, Goding 1981, Robinson et al. 1981b). Tli6 immunization giving rise to the monoclonal anti-fi2M alloantibody, provisionally called mc-/~2M-B :clone 23-IgG2a(~c), described in this report, was SJL/J anti-B10.S spleen and thymus. The mc-fl2M-B reagent used was pooled serum and ascites from pristane-primed (BALB/c x SJL/J)F t mice carrying clone 23 hybridoma in passage. In the cytotoxicity assay with guinea pig serum as complement (rabbit complement gave a somewhat higher titer but a lower proportion of lysed cells), the strain distribution shown in Table 1 accords without exception to the strain distribution of Ly-ml 1 given by Tada and co-workers (1980); the Ly-ml 1 phenotype of the BXD-6 RI strain was originally reported to be Ly-ml 1 negative, apparently because of an error in identification of the tested mice, and is now known to be Ly -ml l positive (U. H~immerling, personal communication). Also, the cytotoxicity activity is in agreement with fl2M-B distinguished by SDSPAGE as described (Michaelson 1981). The proportions of positive cells was > 95~o for LNC, ~ 85~ for spleen, 40~ for bone marrow, and ~ 10~ for thymus, roughly conforming to the comparative sensitivities of these four populations to H-2 antibody in cytotoxicity assays, to a titer of 1 : 640. The bands precipitated by the mc-fi2M from labeled spleen cells are shown in Figures 1 and 2.