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Rabbits preferentially utilize only one of their multiple functional germline immunoglobulin VH genes. This preferential usage of one gene, VH1, raises the question of how rabbits generate antibody diversity. VDJ diversification was analyzed by cloning and sequencing VH1 gene rearrangements. Comparison of these sequences with that of germline VH1 identified clusters of nucleotide changes, including codon insertions and deletions. To investigate whether gene conversion was involved in this somatic diversification, we searched a data base of rabbit germline VH gene sequences for donor VH genes; potential donors were identified for five diversified regions. We conclude that somatic gene conversion has a major role in generating antibody diversity in rabbits. These studies provide clear evidence for somatic gene conversion of mammalian VDJ genes.

Previous experiments have resulted in the identification and cloning of 13 nonallelic genes encoding the constant region of rabbit IgA H chains. The genes, C alpha 1 to C alpha 13, were each cloned into an expression vector containing the VDJ gene of a dansyl (DNS)-binding murine hybridoma and the constructs were then transfected into SP2/0 cells that were producing murine kappa-L chains from the DNS-binding hybridoma. Of the 13 resulting transfectomas, 12 were shown, by ELISA, to secrete DNS-binding chimeric rabbit-mouse IgA molecules. These transfectoma antibodies, representing 12 different isotypes, are of high affinity and provide a unique source of Ag-specific IgA for comparison of the functions of the multiple IgA isotypes. One such function for antibodies is activation of C by either the classical or alternative pathway. We have used the DNS-binding IgA transfectoma antibodies in C assays based on binding of rabbit C3 to IgA-Ag complexes in an ELISA. The results demonstrated that all 12 IgA isotypes are capable of activating C by the alternative pathway but that none can activate C by the classical pathway. Control experiments demonstrated that activation was hapten dependent and was not caused by endotoxin contamination. These data demonstrate that Ag-specific IgA molecules, unmodified by heat or chemical aggregation, activate C by the alternative pathway but not by the classical pathway.

A unique feature of rabbit Ig is the presence of VH region allotypic specificities. In normal rabbits, more than 80% of circulating immunoglobulin molecules bear the VHa allotypic specificities, al, a2 or a3; the remaining 10% to 20% of immunoglobulin molecules lack VHa allotypic specificities and are designated VHa-. A mutant rabbit designated Alicia, in contrast, has predominantly serum immunoglobulin molecules that lack the VHa allotypic specificities (Kelus and Weiss, Proc. Natl. Acad. Sci. USA 1986. 83: 4883). To study the nature and molecular complexity of VHa- molecules, we cloned and determined the nucleotide sequence of seven cDNA prepared from splenic RNA of an Alicia rabbit. Six of the clones appeared to encode VHa- molecules; the framework regions encoded by these clones were remarkably similar to each other, each having an unusual insertion of four amino acids at position 10. This insertion of four amino acids has been seen in only 2 of 54 sequenced rabbit VH genes. The similarity of the sequences of the six VHa- clones to each other and their dissimilarity to most other VH genes leads us to suggest that the VHa- molecules in Alicia rabbits are derived predominantly from one or a small number of very similar VH genes. Such preferential utilization of a small number of VH genes may explain the allelic inheritance of VH allotypes.

Rabbits are unique in that their immunoglobulin VH regions bear allotypic markers encoded by allelic genes. The presence of these markers on most serum immunoglobulins is difficult to explain, as the germline contains several hundred VH genes. We cloned VH genes from normal rabbits of the VHa allotypes a1, a2, and a3 and from a mutant a2 rabbit, Alicia, which expresses almost no a2 allotype. The D-proximal VH gene VH1 of normal rabbits encoded prototype a1, a2, or a3 allotype VH regions in a1, a2, or a3 rabbits, respectively; VH1 was shown to be preferentially utilized in leukemic rabbit B cells. This VH1 gene was deleted from the germline of the Alicia rabbit. These data suggest that the allelic inheritance of a allotypes results from preferential utilization of VH1 in VDJ rearrangements. We suggest that antibody diversity in rabbit primarily results from somatic hypermutation and gene conversion.

VDJ genes were cloned from leukemic B cells of an a1/a2 heterozygous Emu-cmyc transgenic rabbit. Restriction mapping and nucleotide sequence analysis indicated that one clone, 5C3, had a VHa1-encoding gene segment functionally rearranged to a JH gene segment from the a2 chromosome. This VDJ gene may be the result of a trans recombination between a VH gene on the a1 chromosome and a JH gene segment on the a2 chromosome or, it may be the result of a cis recombination if the a2 chromosome contains VHa1-encoding gene segments.

Previous studies have suggested that the majority of rabbit germ-line VH genes encode molecules that are rarely found in serum or secretory Ig. To examine the repertoire of expressed VH genes, we prepared a cDNA library from splenic mRNA of an alpha 1/alpha 1 rabbit and isolated 10 complete VH-encoding cDNA clones. None of the cDNA clones hybridized to an oligomer that had hybridized to more than 50% of cloned germ-line VH genes. These data indicate that only a subset of germ-line VH genes are used in functional VDJ rearrangements. DNA sequence analysis demonstrated that the 10 cDNA clones contained highly similar VH regions, further suggesting that the repertoire of utilized VH genes is limited. In contrast, the D regions of each of the 10 clones exhibited little similarity to one another, suggesting that the rabbit has a large D region repertoire. We propose that the apparent lack of diversity within the VH segment of VDJ rearrangements is offset by extensive D region diversity.

The rabbit MHC class II DP, DQ, and DR alpha and beta chain genes were transfected into murine B lymphoma cells. The transfected cells expressed R-DQ and R-DR molecules on the cell surface but they did not express the R-DP genes either on the cell surface or at the level of mRNA. Northern blot analyses showed that the R-DP genes were expressed, albeit at low levels, in rabbit spleen. Similar analyses showed that the R-DQ and R-DR genes were expressed at high levels in rabbit spleen. A new monoclonal anti-rabbit class II antibody, RDR34, has been developed and shown to react with the R-DR transfected cells and not with the R-DQ transfected cells. The previously described monoclonal anti-rabbit class II antibody, 2C4, reacted with the R-DQ transfected cells and not with the R-DR transfected cells. Thus, 2C4 and RDR34 MAb's are specific for the R-DQ and R-DR molecules, respectively. Each of the antibodies reacted with approximately 50% of rabbit spleen cells as shown by immunofluorescent antibody studies.

Polyclonal B cell leukemias have been generated in 17- to 20-day old Emu-myc transgenic rabbits. To analyze the repertoire of VH genes utilized in early B cells, eight VDJ genes were cloned from these leukemic cells. The nucleotide sequences of these genes indicated that seven of the eight VDJ genes encoded prototype VHa1, VHa2 or VHa3 allotypes. The two VDJ genes encoding VHa1 molecules had VH segments with identical nucleotide sequences; similarly, the VH segments of the four VDJ genes encoding VHa2 molecules were identical, with the exception of a single base pair. These data suggest that a limited repertoire of VH genes were utilized in the generation of these VDJ genes. The DH segments of these genes were limited to two DH families, D1 and D2, indicating that a restricted repertoire of DH genes also had been utilized. Since these leukemic cells probably developed early in ontogeny, we suggest that this restricted utilization of VH and DH genes is representative of B cells from developmentally immature rabbits.

Reprinted with permission from Medicine and Science in Sports and Exercise (American College of Sports Medicine) 17:6 646-650, 1985. The Daily Adjustable Progressive Resistive Exercise (DAPRE) technique was developed clinically in an effort to provide an objective means of increasing resistance concurrently with strength increases during knee rehabilitation subsequent to injury/surgery. The key to the DAPRE technique is that on the third and fourth sets of exercise the patient performs as many repetitions as possible. The number of repetitions performed during the third and fourth sets is used to determine the amount of weight that is added to (or sometimes removed from) the working weight for the next set and session, respectively. Consequently, patients exercise nearer their optimal capacity during each weight rehabilitation session, and their strength redevelopment occurs at a much faster rate. This report describes the quadriceps muscle strength gains by 21 athletes who used the DAPRE technique following knee immobilization for a minimum of 3 weeks. These patients averaged an increase of 4.3 +/- 2.2 (SD) kg.day-1 for a period of 6.4 +/- 2.2 days, as measured by a six repetitions maximum test. It seems unlikely that morphological changes were responsible for these strength increases. It is postulated that strength redevelopment following immobilization involves changes in neural pathways and/or overcoming possible neural inhibitors. J Orthop Sports Phys Ther 1990;12(2):66-71.

Three rabbits of genotype a1n81f73g74/a2n82f71g75 which had been injected at birth with anti-a l (VH) antiserum and which were previously shown to be suppressed for the paternal allotypes a 1, n81, f73 and g74 at 8 weeks of age, were monitored over a 2-year period for the concentration of suppressed and nonsuppressed allotypes in their sera. In all three suppressed animals, the f73 (C alpha) and g 74 (C alpha) allotypes were expressed again at a much greater rate than the a1 (VH) and n81 (C mu) allotypes. In one suppressed animal, the a l (VH) allotype was re-expressed at a much greater rate than the n81 (C mu) allotype and reflected primarily the reappearance of a l IgG. Thus, the escape from allotype suppression in this animal was in the order IgA, IgG, IgM which is the reverse of the order of appearance of these Ig classes during ontogeny. While the al(VH) and n81 (C mu) allotyes remained suppressed, the f73 (C alpha) and g74 (C alpha) allotypes were re-expressed to the same concentration as in the unsuppressed controls, and no compensatory decrease of the f71 and g75 allotypes occurred. During the re-expression of the f73 and g74 allotypes, the ratio of the concentrations of f73/g74 remained approximately constant.

Alloimmunizations with rabbit lymphoid cells have resulted in the identification of two cell-surface alloantigens, Ia1 and Ia2. These antigens reside on nearly all B cells; few, if any thymus cells or T cells of mesenteric lymph nodes bear these antigens. Genetic studies showed that Ia1 and Ia2 molecules appear to be controlled by allelic genes at a locus closely linked to the MHC. Immunochemical analyses revealed that Ia1 and Ia2 are glycoproteins and that each is composed of two polypeptide chains of molecular weights of 28 000 and 30 000-32 000. Thus, the alloantigens identified by these two antisera appear to be Ia-like molecules.

The reaction of sIgA with 5,5'dithiobis (2-nitrobenzoic acid) or with 14C-iodoacetamide revealed an average of 1 mol -SH per mol sIgA under nondenaturing conditions, whereas an average of 7.5 mol -SH was found under denaturing conditions. These -SH groups were found in both the sIgA-f and sIgA-g subclasses; in the sIgA-g subclass, the free -SH groups were found in the Fab alpha portion but not in the Fc alpha portion of the molecule. To determine whether the noncovalently bound polypeptide chains in sIgA contained free -SH groups, sIgA was alkylated with 14C-iodoacetamide and subjected to gel filtration in 6 M guanidine-HCl. This treatment liberated several different polypeptides that were identified as secretory component (SC), L chain dimers, and L chain monomers; measurement of radioactivity incorporated in each of these noncovalently bound chains showed that SC and the L chain dimers did not contain free -SH groups but that an average of 1 -SH group per L chain monomer was found. Of the covalently bound polypeptide chains, J chain and SC did not have detectable -SH groups, whereas the L chains and alpha-chains had 0.3 and 1.2 mol -SH per mol protein, respectively. The identification of free -SH groups in the Fab portion of the sIgA-g subclass and their preferential localization within only some of the L chains reflect the structural heterogeneity of sIgA; these -SH groups presumably arise during biosynthesis.

Germline genes encoding C mu, C gamma, C alpha, and C epsilon heavy chains of rabbit immunoglobulins have been isolated from recombinant phage and cosmid libraries. The JH, C mu, C gamma, and C epsilon are found in a 5'-JH-C mu-C gamma-C epsilon-3' orientation on a 90kb stretch of DNA. Four C alpha genes have been cloned and presumably reside 3' to the other CH genes. Southern blot analysis of rabbit sperm DNA indicates that the rabbit genome contains a single C gamma gene, one C mu gene, and as many as 10 C alpha genes. Restriction site polymorphism is found for C mu, C gamma, and C alpha genes of rabbits of various heavy chain haplotypes. The organization of the rabbit CH genes differs from that of mouse and human CH genes in that the rabbit has multiple C alpha genes, whereas mouse and human have one or two C alpha genes, respectively. In addition, mouse and human have four C gamma genes, whereas rabbit has only one C gamma gene. The presence of a single C gamma gene indicates that at least in the rabbits examined, no germline gene encoding latent or unexpected, C gamma allotypes is present. The genetic control of the expression of latent C gamma allotypes is discussed.

Rabbit secretory IgA (sIgA) was digested with trypsin at 37 degrees C for 30 min and four fractions were isolated by gel filtration. These fragments were characterized by ultracentrifugation, and by their antigenic properties as undigested sIgA, Fab and Fe (Two Fc fragments differing in their content of secretory component were obtained.) The IgA-f subclass molecules were resistant to cleavage and were found in the undigested sIgA fraction; the IgA-g subclass molecules were cleaved into Fe and Fab fragments. The g allotypic determinants of IgA-g molecules were found on both the Fc fragments and the Fab fragments. The Fc and Fab fragments obtained from trypsin-digested sIgA were compared by means of antigenic properties and peptide maps with the Fc and Fab fragments obtained from papain-digested sIgA; no differences attributable to the alpha-chain were found. Thus, papain and trypsin cleave the alpha-chain of IgA-g molecules at similar but not necessary identical positions.

The specificity of rabbit receptors for IgA (RFc alpha) was investigated on lymphocytes isolated from the Peyer's patches and spleen. Specifically was determined by the inhibition of IgA rosette formation with intact and proteolytic fragments of rabbit and human secretory IgA (SIgA), human myeloma proteins, and secretory component. RFc alpha bound human SIgA as efficiently as rabbit SIgA. Of the human IgA tested, RFc alpha preferentially bound to the IgA2 subclass and most avidity to a multimeric IgA molecule. In addition., the site of the RFc alpha-IgA interactions was localized to the C alpha 2 domain in the Fc portion of IgA. RFc alpha on spleen lymphocytes showed the same pattern of binding specificity toward the human IgA subclasses as the RFc alpha on lymphocytes from Peyer's patches. However, differences between the spleen and Peyer's patches were observed in the interaction of RFc alpha and the rabbit IgA subclasses. RFc alpha on lymphocytes from the Peyer's patches bound the rabbit IgA-g subclass more efficiently than the rabbit IgA-f subclass, whereas the converse was true for the spleen RFc alpha. Human secretory component inhibited IgA but not IgG or IgM rosette formation as a result of its interaction with lymphocytes. These studies suggest: 1) that there may be two types of RFc alpha expressed by lymphocytes in certain tissues, and 2) that there are two closely associated receptors on the lymphocyte--one specific for secretory component and the other for structures within the C alpha 2 domain of IgA.

Bovine C mu, C gamma, C alpha and C epsilon genes were cloned in an EMBL4 recombinant phage library using rabbit immunoglobulin switch mu (Su) and human C gamma as probes. Restriction mapping and Southern blot analyses of these clones identified one clone which hybridized with rabbit C mu and JH probes. The HG and C mu regions were separated by 6 kb of DNA. One C alpha and one C epsilon gene were found on overlapping clones and were separated by approximately 15 kb of DNA. Southern blot analysis of germline DNA with a bovine C alpha associated probe (S alpha) indicated that the germline contains a single C alpha gene. Similar analyses with a bovine C epsilon probe indicated that the germline contains either one C epsilon gene with allelic restriction polymorphism or two C epsilon genes. Three C gamma genes were cloned and did not overlap with one another. Southern blot analyses of germline DNA with a bovine C gamma probe indicated that the germline contains a total of four C gamma genes. The genes cloned correspond to three of the four genes identified by Southern blot analysis. The orientation of each CH gene was assigned by hybridization with S mu or S gamma probes. The S gamma probe hybridized to DNA immediately adjacent to all three C genes; the S probe hybridized to DNA immediately adjacent to the C mu, C alpha and C epsilon genes. Unexpectedly, the S mu probe also hybridized with a segment of DNA approximately 7 kb downstream of the C mu gene. This may represent a switch region for C gamma.

The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N3ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the [alpha-32P]8N3ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T31 of the E. coli K-12 RecA protein, which was the unique site of 8N3ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10(7) years.

Heterozygous rabbits of genotype a1n81f73g74/a2n82f71g75 were suppressed at birth for the VH region a1 allotype. At 8 weeks of age, quantitative analysis of serum IgG, IgM, and IgA molecules showed that the VHa1 specificity was effectively suppressed in the three classes of Ig and that the suppression was extended to the CH region n81 specificity on mu-chains as well as to the CH region f73 and g74 specificities on alphaf and alphag chains. At 26 weeks of age, analysis of serum IgG and IgM molecules showed that a1 was still suppressed to approximately the same extent in both Ig classes and the suppression was still extended to the CH region n81 specificity. However, at 26 weeks, the percentage of molecules with a1 specificity had doubled among serum and colostral IgA molecules and this increase was extended to the CH region f73 and g74 specificities. Thus, the suppressed allotypes reappeared first among IgA molecules. Our data are consistent with a regulatory mechanism which controls and synchronizes the expression of the VHa and the CH allotypes expressed on the same heavy chain. The order of the re-expression of the suppressed allotypes with respect to Ig class may allow further definition of selective regulatory mechanisms for the synthesis of Ig classes.

The photoaffinity label 8-azidoadenosine 5'-triphosphate (N3-ATP) was used to covalently modify the recA protein from Escherichia coli within its ATP-binding site. We have previously demonstrated that N3-ATP modification of recA protein is specific for the ATP-binding site and have isolated a unique tryptic peptide (T31), spanning residues 257-280, that contains the exclusive site of attachment of this ATP analog (Knight, K. L., and McEntee, K. (1985) J. Biol. Chem. 260, 867-872). We performed a secondary proteolytic digestion of the [alpha-32P]N3-ATP-labeled T31 peptide using Staphylococcus aureus V8 protease and purified the resulting peptide fragments by high-pressure liquid chromatography (HPLC). Based on a comparison of the amino acid compositions of all purified fragments and sequence analysis of one labeled fragment we determined that Tyr-264 is the exclusive site of N3-ATP attachment in recA protein. Photoaffinity labeling of recA protein was also performed in the presence of single-stranded DNA. Following trypsin treatment and separation of peptides by HPLC we showed that tryptic peptide T31 contained the exclusive site of N3-ATP attachment. A secondary proteolytic digestion was performed on both [alpha-32P]N3ATP-modified T31 and unmodified T31 using alpha-chymotrypsin. Comparison of the HPLC profiles and amino acid compositions of the resulting fragments was consistent with Tyr-264 as the exclusive site of N3-ATP attachment to recA protein.

We have produced a mouse anti-rabbit Ia monoclonal antibody (MAb) that detects an isotypic determinant on all rabbit Ia molecules. This MAb precipitates three polypeptide chains with molecular weights of 28,000, 31,000 and 35,000, corresponding to the Ia beta, Ii and alpha chains, respectively. The anti-Ia MAb inhibits the mixed lymphocyte culture by 80%. In secondary in vitro immune response cultures, the anti-Ia MAb inhibits the proliferative response to bovine insulin and poly (Glu50Tyr50). In studies on mitogenesis it was found that the anti-Ia MAb inhibited the response to LPS but not to concanavalin A or phytohaemagglutinin. The effect of the anti-Ia MAb on other mitogens was found to vary from rabbit to rabbit.

The preferential expression of anti-As antibodies in the allotype a1 of heterozygous a1a3 rabbits immunized with As-TMA-BSA has been investigated by means of quantitative methods. The average content of the anti-As antibodies in a1 and a3 allotypes was 84 and 11%, respectively; the analogous values for anti-TMA antibodies were 41 and 56%, and for anti-BSA antibodies they were 54 and 41%. The molar anti-As/anti-TMA ratios in the heterozygous a1a1 rabbits sensitized with As-TMA-BSA. The very low yields of anti-As-antibodies of allotype a3 cannot be caused by a lack of genes for the production of anti-As antibodies of allotype a3 because a3a3 homozygotes produce considerable amounts of anti-As antibody of allotpye a3. Competition between lymphoid cells having anti-As receptors of different allotype and different affinity for the antigenic p-azobenzenearsonate determinant is discussed as a possible cause for the preferential expression in the a1 allotype.

Screening of recombinant cosmid and phage libraries with a rabbit C alpha cDNA probe has identified a total of seven C alpha genes organized into two clusters; one cluster contains four genes and the other cluster, three genes. Southern blot analysis of genomic DNA indicates that the rabbit genome contains a minimum of ten C alpha genes; at least three genes remain to be cloned. Four of the seven cloned C alpha genes have been tested for their ability to be expressed in vitro. Each gene was cloned 3' to a rearranged murine VDJ gene, and the chimeric gene was then transfected into J558L plasmacytoma cells. Stable transfectants were selected, and each transfectoma was shown to express a chimeric alpha-heavy chain. These chimeric alpha-heavy chains were of two sizes, 55 kd and 60 kd. Each of the four transfectomas secreted chimeric heavy chains in association with endogenous murine lambda light chains. All four C alpha genes tested were expressible, indicating that the rabbit may have multiple IgA subclasses or isotypes. This is in marked contrast to mouse and human which have only one and two subclasses of IgA, respectively.

Rabbit spleen and mesenteric lymph node cells were treated with a monoclonal anti-rabbit T-lymphocyte antibody (MAb) and complement and the effect of the treatment on various lymphocyte functions was determined. Lysis of spleen and mesenteric lymph node cells reactive with this MAb, 9AE10, essentially eliminated their proliferative responsiveness to allogeneic lymphocytes in the mixed lymphocyte reaction and to the T-cell mitogens, concanavalin A and phytohaemagglutinin; responsiveness to the B-cell mitogen, anti-immunoglobulin (Ig) was not decreased by lysis of 9AE10+ cells. In addition, the 9AE10+ cells were found to be necessary for the secondary in vitro antibody response to the T-dependent antigen sheep red blood cells (SRBC), as removal of 9AE10+ cells blocked the generation of plaque forming cells (PFC) in culture. The PFC's themselves were not sensitive to lysis by 9AE10 MAb and complement Thus, the 9AE10 MAb appears to recognize cells which have functions characteristic of T lymphocytes and this monoclonal antibody will be useful in further studies of the rabbit cellular immune system.

Cytoplasmic immunoglobulin of lamina propria plasma cells was analyzed by immunofluorescence on the flow cytometer. Lymphoid cells were made permeable to immunofluorescent reagents by treatment with Triton X-100. These cells were then reacted with FITC (green) anti-light chain and with phycoerythrin (red) anti-heavy chain antibodies. Flow cytometric analysis of these cells revealed that plasma cells expressing cytoplasmic immunoglobulin light and heavy chains were specifically stained by the immunofluorescent reagents. These plasma cells were also shown to have membrane Ig as detected by cell surface immunofluorescence. Light scatter analysis indicated that these plasma cells could be distinguished from lymphocytes by 90 degrees light scatter. These studies provide a method by which several parameters of gut plasma cells can be analyzed by flow cytometry.

This report summarizes our current understanding of the heavy chain haplotypes found in our laboratories' rabbits. Independently derived data from several laboratories have been synthesizes into a consistent picture of the linked inheritance of allotypic markers found on the different heavy chain classes and subclasses of rabbit immunoglobulins in pedigreed rabbits, including the families of three apparent VH-CH recombinants. In one recombinant, the entire group of CH markers (C mu, C gamma, and C alpha) recombined with the set of VH. Although in the other two recombinants all CH markers may also have recombined as a group, in one of these only IgG and IgA CH genes were informative; in the other recombinant, only the IgG allotypes were informative. Some allotypic determinants found on IgM molecules ("conformational") appear only when a specific variable region allotype (VHa) is combined with a specific mu constant region allotype (C mu). New combinations of VHa and C mu allotypes were generated in two of the genetic recombinants and led to new "conformational" determinants. The gains and losses observed lend support to the hypothesis that the determinants result from conformations generated by the combination of allotype-specific VH and C mu protein sequences. Conceivably, DNA events that join VH to diversity (D)- and joining (J)-coding sequences or mRNA processing events that splice J to C mu could be involved in generating the sequences that form allotype-specific determinants.

We have covalently modified the recA protein from Escherichia coli with the photoaffinity ATP analog 8-azido-[alpha-32P]ATP (N3-ATP). Covalent attachment of N3-ATP to recA protein is dependent on native protein conformation and is shown to be specific for the site of ATP hydrolysis by the following criteria. (i) Binding of the probe to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine 5'-O-(thiotriphosphate), ADP, and UTP, but not by adenosine; (ii) N3-ATP is efficiently hydrolyzed by recA protein in the presence of single-stranded DNA; (iii) labeling of recA protein occurs at a single site as judged by two-dimensional thin-layer peptide mapping and high-performance liquid chromatography peptide separation. We have purified and identified a tryptic fragment, spanning amino acid residues 257-280, which contains the primary site of attachment of N3-ATP. This peptide is likely to be contained within the ATP hydrolytic site of recA protein.

Overlapping cosmid clones of rabbit germ-line DNA containing VH, DH and JH gene segments were isolated. The map of this cluster of cosmid clones indicated that the rabbit VH and JH regions were separated by 63 kb. Hybridization of Southern blots of these cosmid clones with two different DH segment probes identified a total of six DH segments within the region between the VH and JH regions. The nucleotide sequences of the JH region and one of the DH segments have been determined. The DH segment has conserved heptamer and nonamer sequences separated by 12 and 11 bp at the 3' and 5' sides, respectively, of the coding region and hence, appears to be a functional gene. The nucleotide sequence of the JH region revealed four functional JH gene segments and one JH pseudogene. Inasmuch as the JH region had previously been linked by contiguous overlapping clones with C mu, C gamma, C epsilon, and one C alpha gene, this VH-DH-JH cluster and the clones containing the Ig H chain C region genes represent 190 kb of contiguous germ-line DNA of the Ig H chain locus.

Based on clinical evidence, cryokinetics (alternating cold and exercise) is replacing heat modalities as the preferred therapy for rehabilitation of traumatic musculoskeletal injuries in athletes. Theories have been advanced to explain the clinical successes of cryokinetics, but little scientific data have been collected. Strain gauge plethysmography was used to measure blood flow to the ankle of 12 uninjured male subjects. A repeated measures design was utilized with each subject being tested under six experimental conditions: 1) heat packs, 2) cold packs, 3) control, 4) heat-exercise, 5) cold-exercise, 6) control-exercise. Exercise consisted of 5 three-minute bouts (3.5 mph) interspersed with heat, cold, or control throughout a 45-minute period. Non-exercise, heat and cold were administered for 25 minutes each, followed by 20 minutes without treatment. Instantaneous blood flow was measured regularly during non-exercise periods, estimated during exercise, and total flow was computed by integrating over the 45 minute treatment-post treatment period. Total flow (ml flow/100 ml tissue/min) was greater/p. less than .0002) during cold-exercise than during heat treatments. Contrary to some theories, there was neither cold-induced vasodilatation during, nor a reflex vasodilatation following, the 25-minute cold application. These data suggest that during cryokinetics, exercise causes the increased blood flow, and that cold applications function only to allow active motion in a painful joint.

Heterozygous rabbits representing 9 of 15 possible a1 and a2 heavy chain haplotype gene combinations among rabbits in the University of Illinois colony were analyzed for ratios of a1 to a2 in serum immunoglobulin (Ig). The Ig from rabbits of the a1x-y-n81f73g74de12,15 heavy chain haplotype in combination with any of three a2-associated heavy chain haplotypes have higher ratios of a1 to a2 than Ig from rabbits in which a1 is encoded by 4 other heavy haplotypes. For example, the mean a1:a2 ratio for adult a1x-y-n81f73g74de12,15/a2x32y33,- n82,f71g75de12,15 rabbits was 12:1 compared to 5:1 for a1x-y33,30n83,f71g75de12,15/a2x32y33,- n82,f71g75de12,15 heterozygous rabbits. Family studies indicated that the a1:a2 ratio was under the control of the heavy chain chromosomal region or a locus closely linked to it. Whether the regulation is due to varying numbers of VH genes and/or J gene segments, a separate regulator gene, or more efficient joining of certain gene segments, has yet to be determined.

Transgenic rabbits with the rabbit c-myc oncogene fused with the rabbit immunoglobulin heavy chain enhancer region (E mu) DNA were developed by microinjecting pronuclei of single cell zygotes with the gene construct and implanting the microinjected eggs into pseudopregnant females. At age 17-20 days, 3 of 21 offspring born to seven females were found to have peripheral blood leukocyte counts of greater than 100,000 per mm3. Histology analyses showed extensive lymphocytic infiltration in the liver and kidney, indicating that these rabbits had a malignant lymphocytic leukemia. Genomic DNA analyses of thymus and peripheral blood lymphocytes revealed that the leukemic rabbits were transgenic and that both immunoglobulin heavy and kappa light chain genes were rearranged in the leukemic cells; thus, the leukemic cells are of B-cell lineage. One to four heavy and light chain gene rearrangements were observed, suggesting that the B-cell tumors were oligoclonal. Stable tissue culture cell lines from the bone marrow and peripheral blood of one of the transgenic rabbits have been developed. The development of B-cell leukemias in rabbits with the E mu-myc transgene contrasts with the development of B-cell lymphomas in mice carrying a similar transgene. The lymphomas in mice develop in adults and are monoclonal in origin. The leukemias in rabbits develop in juveniles, less than 3 weeks after birth, and appear oligoclonal in origin. The leukemias seem to develop in rabbit at a specific stage of development, and we suggest that a normal developmental signal may be involved in the oncogenesis.

The immunoglobulin JHC mu intron was cloned from genomic DNA of a VHa3 rabbit and a 1257 bp sequence which contains conserved enhancer and splice sites was determined. From positions 315 to 1257, there is approximately 72 and 67% similarity to available sequences of man and mouse, respectively (counting gaps as single changes at single positions). In earlier studies of rabbit cDNAs encoding immunoglobulin heavy chains, we found a C mu-encoding cDNA clone (pB3) derived from splenic mRNA of a Trypanosome-hyperimmunized rabbit (VHa1) which lacked VH, DH or JH sequences and had an unknown sequence 5' of that encoding C mu. Comparison of this cDNA sequence with the present cloned genomic DNA sequence has now revealed that the start of cDNA pB3 corresponds to a position 80 base pairs 3' of the conserved octamer motif of the rabbit heavy chain enhancer. This mRNA was spliced to the acceptor site of C mu using a donor site which was 635 bp 3' of the enhancer octanucleotide. Our sequence of pB3 indicates that in rabbit as in mouse, a "nontron" (33 stop codons in three reading frames) can be formed utilizing a conserved splice site to produce a spliced transcript. The presence of evolutionarily conserved splice donor sites in the intron sequences of rabbit, mouse and man suggests a functional role during B cell ontogeny.

The Daily Adjustable Progressive Resistive Exercise (DAPRE) technique was developed clinically in an effort to provide an objective means of increasing resistance concurrently with strength increases during knee rehabilitation subsequent to injury/surgery. The key to the DAPRE technique is that on the third and fourth sets of exercise the patient performs as many repetitions as possible. The number of repetitions performed during the third and fourth sets is used to determine the amount of weight that is added to (or sometimes removed from) the working weight for the next set and session, respectively. Consequently, patients exercise nearer their optimal capacity during each weight rehabilitation session, and their strength redevelopment occurs at a much faster rate. This report describes the quariceps muscle strength gains by 21 athletes who used the DAPRE technique following knee immobilization for a minimum of 3 wk. These patients averaged an increase of 4.3 +/- 2.2 (SD) kg X d-1 for a period of 6.4 +/- 2.2 d, as measured by a six repetitions maximum test. It seems unlikely that morphological changes were responsible for these strength increases. It is postulated that strength redevelopment following immobilization involves changes in neural pathways and/or overcoming possible neural inhibitors.

A non-IgM immunoglobulin molecule was found on most rabbit Ig-bearing lymphocytes isolated from mesenteric lymph nodes. Membrane bound immunoglobulin light chains and heavy chains were detected by immunofluorescence and by rosetting with antibody-coated erythrocytes on mesenteric lymph node cells stripped of IgM by anti-IgM allotype antibodies. The percentage of cells bearing these residual immunoglobulin molecules was similar to the percentage of cells bearing immunoglobulin before "stripping" with anti-IgM antibody. These residual immunoglobulin molecules were not IgA nor IgG and are believed to be the rabbit analogue of human IgD.

Two rabbit germ-line VH gene segments have been isolated from a recombinant phage DNA library. Nucleotide sequence analysis indicates that both of the genes share structural and regulatory features common to mouse and human VH genes, although one appears to be a pseudogene. Comparison of the protein sequences encoded by these genes to the protein sequences of rabbit immunoglobulin V regions indicates that both genes encode VH a-negative-like molecules. Quantitative genomic blot analysis with a VH probe capable of recognizing most, if not all, germ-line VH genes indicates that there are approximately 100 VH genes in the haploid genome of rabbits. The average spacing between the germ-line VH genes was determined to be approximately 6 kb. The molecular basis for the allelic inheritance of rabbit VH allotypes is discussed in view of the structural organization of germ-line VH genes.

A cDNA library was constructed by using rabbit spleen poly(A)+RNA as template, and from this library was isolated a cDNA clone, p2a2, that encodes 179 amino acids of the heavy chain of rabbit IgG. The nucleotide sequence of p2a2 showed that it encodes the COOH-terminal eight amino acids of the CH1 domain, the hinge region, the CH2 domain, and the NH2-terminal half of the CH3 domain of C gamma. Southern blot hybridization analysis of rabbit sperm DNA showed that two EcoRI fragments hybridized strongly with the C gamma cDNA. The p2a2 cDNA was used as a probe to isolate recombinant Charon 4A phage clones containing C gamma sequences from a genomic library of rabbit liver DNA. Two distinct DNA segments were identified by restriction mapping and hybridization analysis, suggesting that the haploid rabbit genome may contain two different C gamma genes.

Genetic, biochemical, and biophysical studies have begun to reveal details of the structures of Arc and Mnt and show that these repressors use residues at their N-terminal ends for operator recognition and binding. Some of the DNA contacts made by these residues have been identified, and this information together with NMR studies has permitted the construction of models of the DNA binding region. Although the accuracy of these models remains to be determined, it seems clear that Arc and Mnt are members of a new class of DNA-binding proteins.

The Arc and Mnt repressors of phage P22 are related proteins that bind to different operator DNA sites. By creating a hybrid Arc-Mnt protein, we show that the binding specificity of Mnt can be switched to that of Arc by replacing six residues at the N terminus of Mnt with the corresponding nine residues from Arc.