Structural identity of the TL antigen homologues derived from strain 2 and strain 13 guinea pigs

Strain 2 and strain 13 guinea pig thymocytes have been shown to bear a molecule that by several criteria appears to be a homologue of the murine TL antigen. The existence of a TL polymorphism in the mouse system as evidenced by TL- strains and various TL phenotypes in TL+ strains prompted a study to determine if a similar polymorphism could be demonstrated in the guinea pig system. By using two-dimensional gel electrophoresis, the thymocytes of a third inbred strain, DHCBA, were shown to bear a TL antigen, and the TL antigens of strains 2 and DHCBA were shown to give identical patterns of spots. A biochemical comparison of the strain 2 and strain 13 TL antigen heavy chains by tryptic and chymotryptic peptide mapping demonstrated that these molecules have identical peptides. Thus, no polymorphism could be demonstrated within the guinea pig TL system for the three inbred strains studied. Comparative tryptic peptide mapping of the guinea pig TL and class I B.1+S antigens demonstrated 43% homology, significantly higher than that reported for murine H-2 and TL antigens. These results provide suggestive evidence that the gene duplication giving rise to the genes determining the class I and TL antigens may have occurred more recently in the guinea pig than in the mouse.     

Structural polymorphism of murine factor B controlled by a locus closely linked to the H-2 complex and demonstration of multiple alleles

New alleles of murine factor B (Bf) protein were demonstrated. When ethylenediaminetetraacetic acid (EDTA)-plasmas from inbred and wild mice were analyzed by isoelectro-focusing (IEF) and immunofixation, murine Bf proteins were visualized as distinct protein bands in all mice tested. Four variants of murine Bf could be demonstrated in a large number of tested mice: Bf 1 (isoelectro-focusing point (P.I.) range of 5.8–6.1) exemplified by B10 and B10.BR, Bf 2 (P.I. range of 5.8–6.0) exemplified by B10.MOL (OHM), Bf 3 (P.I. range of 5.6–5.9) exemplified by B10.MOL (TEN2) and Mus musculus (Mus m.) subspecies Chc, Bf4 (P.I. range of 6.0–6.3) exemplified by Mus m. subspecies Shh. The genetic linkage between S locus and Bf locus was studied with two backcross progenies — [B 10.BR × (B10.BR × Mus m. subspecies Chc)F₁] and [B 10.BR × (B10.BR × Mus m. subspecies Shh)F₁]. Totally, 256 backcross progenies were typed for Bf type and for Ss type (plasma level of the fourth complement protein regulated by S locus). The results indicated that murine Bf was controlled by a single codominant locus located close to the H-2 complex because no mouse showing recombination between Bf locus and S locus was found.     

Murine factor B: Electrophoretic polymorphism

The electrophoretic behavior of Bf was investigated by the immunofixation procedure, using a locally produced goat antimouse Bf reagent. A single phenotype was found in the serum of mice of 16 inbred strains. Sera from noninbred Swiss-Webster mice, however, were typed as belonging to one of three phenotypes-Bf F, Bf FS, and BF S-almost identical to those found in human sera.     

Ia antigens in inbred rats and their relationship to the MLR phenotype.

Three different alloantisera were raised by using Ag-B/MLR disparate rats, and the cytotoxic activity remaining after absorption with erythrocytes to remove anti-Ag-B antibodies was examined. The alloantisera detected surface antigens present only on B cells and segregation studies demonstrated that the genes that code for these antigenic specificities were linked to the major histocompatibility complex. The reactivity of the alloantisera with splenic lymphocytes from a panel of strains representative of the currently known Ag-B groups showed that multiple specificities were present in two of the three antisera and that these specificities were shared by many inbred strains. The appropriate absorption studies showed, however, that each antiserum detected an unique specificity that was found only in those inbred strains that shared the same mixed lymphocyte reactivity (MLR) phenotype as the donor strain. The alloantiserum produced against the KGH strain inhibited the MLR reactions involving this strain only when it was used as the stimulating cell population. The antigens detected by the three alloantisera described here have the characteristics of Ia antigens, and they have tentatively been designated Ia.1 (ACI anti-KGH), Ia.3 (B3 anti-BN) and Ia.4 (MNR anti-DA).     

Second genetic polymorphism of tear proteins in the rat (Rtp-2)

Electrophoresis of tear proteins on agarose gel showed polymorphism in the fastest migrating protein among 32 inbred strains of rats. In 8 strains, the protein was missing (RTP-2 B), while the other strains expressed the protein (RTP-2 A). The trait was found to be controlled by a single autosomal locus. The designation Rtp-2 locus, with two alleles (Rtp-2a, Rtp-2b), is tentatively proposed. The Rtp-2 locus is loosely linked to c locus, with a recombination frequency of 36.7 +/- 5.4 percent.     

Peptide mapping analysis of the avian progesterone receptor

Progesterone receptor from the chicken oviduct has been shown to exist as two 8 S forms (I and II). Form I contains a protein of Mr = 75,000 and form II contains a protein of Mr = 110,000. In addition to these hormone-binding proteins, both receptor forms contain a protein with Mr = 90,000 that does not bind steroid. To investigate the possibility that these proteins are structurally related, they were isolated by preparative sodium dodecyl sulfate gel electrophoresis and subjected to peptide mapping analyses after digestion with Staphylococcus aureus V-8 protease, papain, or alpha-chymotrypsin. Receptor proteins labeled with [32P]orthophosphate in tissue minces were also subjected to peptide mapping analysis. The electrophoretic patterns of peptide fragments of the 90-kDa protein from receptor forms I and II were identical but were different from the peptide patterns obtained from the 75- and 110-kDa proteins which generated similar peptide patterns, indicating that these are structurally related. However, some differences were evident, indicating that these latter two proteins are not identical substrates for proteases. A one-dimensional comparison of the phosphopeptide patterns from the 75- and 110-kDa proteins also showed them to be similar, but not identical. Two-dimensional maps of phosphopeptides generated from the 75- and 110-kDa protein after complete tryptic digestion revealed multiple sites of phosphorylation which were identical except for one phosphopeptide that was unique to the 110-kDa protein. These results show the two progesterone-binding proteins to be very similar in structure, but to differ considerably from the 90-kDa protein.     

Genetic control of lipid transport in mice. II. Genes controlling structure of high density lipoproteins

Genetic factors controlling the structure of high density lipoproteins (HDL) in mice have been examined. Surveys of inbred strains of mice revealed genetic structural variations of the two major apolipoproteins of mouse HDL, apolipoproteins A-I and A-II. The structural variations alter the charge of the proteins as judged by isoelectric focusing of HDL under denaturing conditions. The structural variations are inherited as single Mendelian genes exhibiting co-dominant expression. The structural gene for mouse apolipoprotein A-II, designated Alp-2, resides on mouse chromosome 1, tightly linked to Ly-m20, a lymphocyte alloantigen locus. Previous studies, as well as our results, suggest that the structural gene for mouse apolipoprotein A-I, designated Alp-1, is on mouse chromosome 9. The genetic structural variation for apo-A-I results in a shift in the charge of the entire family of apo-A-I isoforms, indicating that they are all encoded by a common structural gene. The structure of intact HDL, examined primarily by electrophoretic techniques, exhibits numerous and complex phenotypes among different strains of mice. One variation, controlling the density and possibly the size of HDL, has been studied in two sets of recombinant inbred strains of mice. The results indicate that the variation is controlled by a single major gene that is either tightly linked to or identical with the Alp-2 gene on chromosome 1. In addition to structural variation, inbred strains of mice exhibited considerable quantitative variation of plasma HDL. Thus, the mouse provides a useful model system for examining the genetic control of mammalian HDL structure and regulation.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. IV. Partial tissue distribution and mapping of immunodominant antigens

The immunization of C57BL/6 responder mice with spleen cells from H-2-matched BALB.B donors, which differ by multiple non-H-2 histocompatibility (H) antigens, results in the generation of cytotoxic T lymphocytes (CTL) that are specific for only a limited number of immunodominant antigens. Previous analysis of the genes encoding these dominant antigens has not mapped these genes to any of the non-H-2 H loci defined by congenic strains. It would have been expected that the histogenetic techniques employed for congenic strain selection would have preferentially identified the "strongest" H antigens. Therefore, we have investigated the possibility that immunodominant antigens do not belong to the class of non-H-2 H antigens encoded by genes mapping to H loci defined and mapped by congenic strains. The first experiments were aimed at identifying antigens that were expressed by independently derived inbred strains and were cross-reactive with the immunodominant cytotoxic T cell target (CTT-1) antigen of BALB.B. Strong cross-reaction with the C3H.SW (H-2b) strain was observed; the C3H gene encoding this antigen was mapped with BXH recombinant inbred strains. Contrary to the mapping of the CTT-1 gene to chromosome 1 in BALB.B, the C3H gene was shown to map to either chromosome 4 or chromosome 7. This result indicates that identical, or at least extensively cross-reactive, non-H-2 antigens may be encoded by genes mapping to independently segregating loci in different inbred strains. The tissue distribution of immunodominant antigens was approached by determining the reactivity of CTL specific for these antigens with either lymphoid-derived or fibroblast-derived targets. These CTL effectively lysed lymphoblast and lymphoid tumor targets but did not lyse an SV40-transformed fibroblast line that was shown to be efficiently lysed by CTL specific for non-H-2 H antigens defined by congenic strains. Therefore, it was concluded that immunodominant antigens detected by B6 anti-BALB.B CTL have a restricted tissue distribution in comparison to non-H-2 H antigens defined by congenic strains. The implications of these results for our understanding of the origin and heterogeneity of non-H-2 cell-surface antigen recognized by effector T cells are discussed.     

The genetics of glutamic-pyruvic transaminase in mice: Inheritance,electrophoretic phenotypes,and postnatal changes

Glutamic-pyruvic transaminase (GPT, E.C. 2.6.1.2) from 18 inbred strains of mice was subjected to starch gel electrophoresis. Two electrophoretic phenotypes were observed: a fast-migrating pattern in 16 strains and a slower-migrating pattern in two strains. A comparison of electrophoretic patterns of F₁ and backcross progeny of two strains of mice showed that the inheritance of GPT is autosomal with two codominant alleles. The genetic locus for GPT is designated Gpt-1, and its two alleles are designated Gpt-1 ^a and Gpt-1 ^b to represent the fast-migrating (A) and slow-migrating (B) patterns. The GPT was expressed in 11 tissues with different amounts of enzyme activity. Developmental studies of GPT activity in liver showed that between 5 and 12 days after birth the mean activity was 10 units/g protein. Between 12 and 19 days, a dramatic rise in activity occurred and adult values of 300 units/g protein were reached by 26 days.This research was supported by The National Foundation (CRBS-258) and the National Institutes of Health (GM15253).Preliminary results were reported at the Annual Meeting of the American Society of Human Genetics, October 11–14, 1972, in Philadelphia.R. P. D. is an investigator of the Howard Hughes Medical Institute.     

Identification and characterization of a porcine parvovirus nonstructural polypeptide.

Sera from porcine parvovirus (PPV)-infected swine fetuses immunoprecipitated and 84- to 86-kilodalton polypeptide in addition to the A and B virion structural proteins. This polypeptide, designated NS-1, was present in PPV-infected cell lysates but not in purified virions. Partial proteolysis mapping revealed that NS-1 was not related to the A and B viral structural proteins. All three proteins in infected cells were phosphorylated at serine residues, and NS-1 also contained phosphothreonine. From pulse-labeling experiments with either 32Pi or [35S]methionine, NS-1 was found to first appear 5 to 7 h postinfection, whereas the viral structural polypeptides were first synthesized 9 to 11 h postinfection. Pulse-chase experiments revealed that NS-1 initially appeared as an 84-kilodalton protein and was subsequently structurally modified to forms of slower electrophoretic mobilities. The time of appearance of NS-1 after virus infection coincided with the initiation of viral DNA synthesis, suggesting that this polypeptide (and the modified forms thereof) may be involved in PPV replication.     

The LEW.BN(2R) strain: a recombinant in the rat MHC

Cell-mediated cytolytic (CMC) responses resulting from immunizations between rat strains considered to be RT1 (Ag-B) identical (LEW.B3:BN) are capable of detecting a membrane determinant(s) controlled by a locus linked to RT1, which has been designated Ag-L. The Ag-L gene region has been isolated in a recombinant line, tentatively designated as LEW.BN(2R), and has been assigned the RT1r5 haplotype. The data presented demonstrate that the genes responsible for MLR stimulation in the 2R strain are of LEW origin. In addition, LEW.B3 anti-BN CTL appear to recognize multiple specificities, only one of which is in the 2R strain. Some of the remaining specificities in BN may be the result of interactions between undetected genes that have been separated in the LEW.B3 and 2R strains.     

Inheritance of mitochondrial DNA in the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>

By crossing Brachionus plicatilis s.s. NH1L strain and German strain, we obtained two types of hybrids, NH1L female × German male designated as NXG and German female × NH1L male designated as GXN. To confirm the crossing of the two hybrid strains at the genetic level, random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis using 10 kinds of primers (10 and 12 mers) was carried out. Some amplified DNA fragments from RAPD of the hybrid strain showed mixed patterns of both parental strains, thus confirming that both hybrids were crossbreeds of the NH1L and German strains. Using these hybrids, we investigated the mode of mitochondrial inheritance in B. plicatilis. Full-length mtDNA of the four strains was amplified by PCR, and digested with restriction enzymes to obtain restriction fragment length polymorphism (RFLP) patterns. Both hybrid strains had the same RFLP patterns as their female parents. This result shows that mitochondrial inheritance in rotifers is maternal. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont and R. Rico-Martínez Advances in Rotifer Research     

Close linkage between mouse genes determining the two forms of complement component C6 and component C7, and cis action of a C6 Regulatory Gene

Two forms of mouse complement component C6, with molecular weights (M _rs) of 90 and 100 kilodaltons (kd), are present in the sera from certain inbred strains such as the CBA strain; other strains, such as the BALB/c and DBA/2 strains, have only the 90 kd C6A form. The present work was undertaken to determine whether the two M _r forms were the products of genes coding at separate loci. We screened sera from mice from a number of inbred strains by isoelectric focusing and found one strain, AKR, exhibiting allotypic structural variations of C6 forms. To distinguish the various types, we designated the 90 kd types from CBA and AKR mice C6A1 and C6A2, respectively, and the corresponding 100 kd types C6B 1 and C6B2, respectively. Mice possessing only one M _r form were all typed as C6A1. Results of breeding experiments strongly suggested that the two M _r forms of C6 are coded for at two closely linked loci. Sera from a number of inbred strains were also screened for a complement C7 polymorphism by means of isoelectric focusing and functional overlay. C7 from all strains, excepting the AKR strain, produced identical C7 band patterns. AKR C7 produced a unique band pattern, and results of breeding experiments with AKR and BALB/c mice showed the C6 and C7 loci to be closely linked. In addition, we identified a regulatory gene for C6 production. The gene apparently requires androgen to facilitate C6 production in the majority of strains. In these strains C6 activity is virtually absent from female sera. However, we observed moderate levels of C6 activity in sera from IS/Cam females, indicating that, in this strain, male physiological androgen levels are not necessary for C6 production. IS/Cam possess one form of circulating C6 which appears identical with BALB/c C6A1, and therefore IS/Cam mice differ from AKR mice at both the C6 structural and regulatory loci. These two strains were thus suitable for use in breeding experiments to determine the manner of action of the regulatory gene. Results showed that it acted in a cis manner.Abbreviations used in this paper M _r molecular weight - kd kilodaltons - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - IEF isoelectric focusing - Slp sex-limited protein - MHC major histocompatibility complex     

Five structural classes of major outer membrane proteins in Neisseria meningitidis

Group B Neisseria meningitidis is thus far subdivided into 15 protein serotypes based on antigenically different major outer membrane proteins. Most serotypes have three or four major proteins in their outer membranes. Comparative structural analysis by chymotryptic 125I-peptide mapping was performed on these major proteins from the prototype strains as well as from six non-serotypable strains. The major outer membrane proteins from each of the serotypes were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the Laemmli system. Individual proteins within the gel slices were radioiodinated and digested with chymotrypsin, and then their 125I-peptides were separated by electrophoresis and chromatography on cellulose thin-layer plates. The peptide maps obtained by autoradiography were categorized into five different structural classes which correlated with the apparent molecular weights of proteins, i.e., 46 +/- 1K, 41 +/- 1K, 38 +/- 1K, 33 +/- 1K, and 28 +/- 1K. Each of the major outer membrane proteins within a strain had a distinctly different chymotryptic peptide map, indicating significant differences in the primary structure of these proteins. In contrast, outer membrane proteins of the same or very similar molecular weight from different serotype strains had similar, occasionally identical peptide maps, indicating a high degree of structural homology. The unique peptides from proteins of the same structural classes were often hydrophilic, whereas common peptides were often hydrophobic, suggesting that the serotype determinants reside within the variable hydrophilic regions of major outer membrane proteins.     

Major histocompatibility complex-linked control of the murine immune response to myelin basic protein

Recent experiments have shown that different regions of myelin basic protein (MBP) are encephalitogenic for different inbred strains of mice. It was therefore of interest to determine whether the immune response to MBP was MHC associated, and if so, what subregion controlled this response. Because PL/J and A/J mice were good responders to mouse MBP and C57Bl/10SN were not, B10.PL(73NS) and B10.A mice were immunized with mouse MBP under conditions designed to induce EAE. These strains were found to be highly susceptible. Intra-H-2 recombinant mice were then assessed for susceptibility. B10.A(4R) and B10.MBR were susceptible, whereas B10.A(5R) were resistant. Thus, EAE induced by purified MBP is under the control of the MHC, and the response maps to the I-A subregion. Production of IL 2 in vitro by T cells from MBP-primed mice in the presence of antigen and adherent cells was blocked by monoclonal antibody to the I-A, but not the I-E, subregion. When the specificity of the encephalitogenic response was tested, peptide 1-37 was active in B10.PL(73NS) and B10.A mice, whereas peptide 89-169 was active in A.SW, SWR, and B10.T(6R) strains. Serum from mice immunized with MBP peptides was assayed for antibody content. PL, B10.PL, and B10.A mice made a good antibody response to peptides 1-37 and 43-88 but were nonresponsive to peptide 89-169. SJL, A.SW, SWR, and B10.T(6R) mice responded well to peptide 89-169 but were poorly responsive to peptides 1-37 and 43-88.     

Heterogeneity of S-layer proteins of Lactobacillus acidophilus strains.

An S-layer (surface regular array) was found in the cell wall from six out of ten strains of Lactobacillus acidophilus examined by electron microscopic observations. All of the six strains which were shown to carry the S-layers belonged to the deoxyribonucleic acid (DNA) homology group A, but not to B, which had been classified by Johnson et al (Int. J. Syst. Bacteriol. 30: 53-68, 1980). On the other hand, the other four strains which possessed no S-layers were in the homology group B. The apparent molecular weights of the S-layer proteins ranged from 41 to 49 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the S-layer proteins from the six strains, three were susceptible to chemical cleavage with N-chlorosuccinimide, giving different peptide maps. All of the six S-layer proteins were fragmented by limited proteolysis with Staphylococcus aureus V8 protease, and gave markedly different peptide patterns by the subsequent peptide mapping analysis, except that the peptide maps of the S-layer proteins from the two strains which were in the same subgroup were identical.     

Structural analysis of hemolymph proteins from Schistosoma mansoni (Trematoda)-susceptible and resistant Biomphalaria glabrata (Gastropoda)

1. Five different molecular weight polypeptides from serum (cell-free hemolymph) of Schistosoma mansoni-resistant and susceptible strains of Biomphalaria glabrata, were examined by two-dimensional 125I-peptide mapping and high performance liquid chromatography (HPLC). 2. Peptide mapping indicated that all five radiolabeled polypeptides within and between the two snail strains had similar migration patterns when cleaved with pepsin or alpha-chymotrypsin, thus revealing a shared structural homology. All peptides chosen for analysis appeared to be structurally similar to the 160 kDa hemoglobin molecule. 3. Separations of the radiolabeled enzyme digests by HPLC confirmed results seen in the mapping experiments since all chromatograms had similar elution patterns. 4. Minor differences in the peptide maps and chromatograms within and between snail strains may be due to quantitative differences in the amount of protein present and/or variations in the primary amino acid sequences of the proteins chosen for analysis.     

Ability of nonpermissive mouse cells to express a simian virus 40 late function(s)

Mouse cells are fully nonpermissive for simian virus 40 (SV40). Infection does not lead to detectable virus replication. In this report, it was shown, first, that spliced 16S and 19S SV40 late mRNA were present in cytoplasmic and polysomal polyadenylated acid+ RNA preparations from SV40-infected baby mouse kidney cells. The 16S and 19S SV40 late mRNA's produced in infected baby mouse kidney cells were identical to or similar to the 16S and 19S SV40 late mRNA's produced in permissive monkey cells as judged by their S1 mapping patterns performed with the late strand of HpaII-BamHI fragment B and by their sedimentation patterns in a sucrose gradient. It was also shown that the 16S late mRNA from infected baby mouse kidney cells could be translated into a polypeptide which was identical to or similar to virion protein VP1 in every aspect examined, including the patter of peptide mapping by limited proteolysis. Second, we reported that mouse kidney cells produced detectable, although low, levels of SV40 virion protein VP1, as shown by the sodium dodecyl sulfate-polyacrylamide gel autoradiogram of [35S]methionine-labeled proteins immunoprecipitated by a rabbit antiserum directed against SV40 virion proteins. Third, it was reported that infected baby mouse kidney cells produced late mRNA's either (i) when the infection was done at a restrictive temperature with the nonleaky tsA58 mutant or (ii) in cells treated with 100 microgram of cycloheximide per ml, in which large T antigen synthesis was inhibited by more than 99.9%. This suggested that large T antigen was not required for the synthesis of late mRNA in mouse cells.     

Characterization, chromosome assignment, and segregation analysis of endogenous proviral units of mouse mammary tumor virus.

In the course of analyzing sites of proviral integration in tumors induced by mouse mammary tumor virus (MMTV), we have isolated recombinant DNA clones corresponding to the 5' and 3' ends of four endogenous MMTV proviruses present in BALB/c and BR6 mice. This has permitted the structural characterization of each locus by detailed restriction mapping and the preparation of DNA probes specific for the cellular sequences flanking each provirus. These probes have been used to trace the segregation patterns of the proviruses, designated Mtv-8, Mtv-9, Mtv-17, and Mtv-21, in a panel of inbred strains of laboratory mice and to map Mtv-17 and Mtv-21 to mouse chromosomes 4 and 8, respectively. The unambiguous resolution of these four proviruses on Southern blots has greatly facilitated the analysis of other endogenous MMTV proviruses in these inbred mice.     

The genetic contribution to heart rate and heart rate variability in quiescent mice

Recent studies have suggested a genetic component to heart rate (HR) and HR variability (HRV). However, a systematic examination of the genetic contribution to the variation in HR and HRV has not been performed. This study investigated the genetic contribution to HR and HRV using a wide range of inbred and recombinant inbred (RI) mouse strains. Electrocardiogram data were recorded from 30 strains of inbred mice and 29 RI strains. Significant differences in mean HR and total power (TP) HRV were identified between inbred strains and RI strains. Multiple significant differences within the strain sets in mean low-frequency (LF) and high-frequency (HF) power were also found. No statistically significant concordance was found between strain distribution patterns for HR and HRV phenotypes. Genomewide interval mapping identified a significant quantitative trait locus (QTL) for HR [LOD (likelihood of the odds) score = 3.763] on chromosome 6 [peak at 53.69 megabases (Mb); designated HR 1 (Hr1)]. Suggestive QTLs for TP were found on chromosomes 2, 4, 5, 6, and 14. A suggestive QTL for LF was found on chromosome 16; for HF, we found one significant QTL on chromosome 5 (LOD score = 3.107) [peak at 53.56 Mb; designated HRV-high-frequency 1 (Hrvhf1)] and three suggestive QTLs on chromosomes 2, 11 and 15. In conclusion, the results demonstrate a strong genetic component in the regulation of resting HR and HRV evidenced by the significant differences between strains. A lack of correlation between HR and HRV phenotypes in some inbred strains suggests that different sets of genes control the phenotypes. Furthermore, QTLs were found that will provide important insight to the genetic regulation of HR and HRV at rest.