Restimulation in secondary MLC by a non-D-locus determinant within the MHC

By testing a family in which one offspring had inherited a chromosome including a recombination between theHLA-B andD loci, we sought to obtain some insight into whether determinants other than those atHLA-D are capable of restimulating in a secondary MLC. Results obtained in the primary MLC indicated that the recombinant child did not express products of theHLA-D region normally associated with this haplotype. When sensitization in a primary MLC was to the entire haplotype, everyone who had the sensitizing haplotype restimulated strongly and specifically. In addition, however, weak to moderate stimulation was obtained when the cells of the recombinant child were used to restimulate. Presumably these cells possessed determinants coded for within theHLA-A toB chromosomal segments of the sensitizing haplotype, but not those coded for by theHLA-D locus. Our results indicate that a simple structure atHLA-D is not the sole factor in the secondary MLC. Either the products of loci outside theHLA-D locus can also restimulate or the recombination occurred within theD locus, suggesting that theD region contains more than one locus coding for restimulating determinants.     

Heterogeneity ofHLA defined in PLT: A cellular assay detects differences not seen using HLA-DRw serology

The primed lymphocyte typing test (PLT) is used to detect the gene products of theHLA-D region which are responsible for secondary restimulation of cells primed in MLC. Alternatively, products of theHLA-D region may be detected serologically using antisera directed against a subpopulation of lymphocytes; these are the so-called DRw determinants. The PLT was used to see if it were possible to detect heterogeneity within a given serologically defined group using a cellular test. As priming combinations, we used family members identical for one haplotype and differing in theHLA-A, B andC regions, but not theD region of the second haplotype. Our results indicated that it was possible to prime against this second haplotype and that the segregation of the difference followedHLA. Therefore, using a cellular test it was possible to detect differences among cells belonging to a given DRw group. This suggests that PLT can be a useful tool to identify those serological groups which are composed of heterogenous determinants. In addition, it points out the problem in using any one test to establish identity of theHLA-D region, especially for clinical purposes.     

Linkage between C2 deficiency and theHLA-A10,B18,Dw2/BfS haplotype in a French family

A linkage between C2 deficiency and the HLA-A10,B18/BfS antigens has been found in a French family from the Strasbourg area. The propositus, suffering from a chronic glomerulonephritis, is homozygous forHLA-A10,B18/BfS and totally C2-deficient. The parents and the brother are heterozygous for C2 deficiency and share theHLA-A10,B18/BfS haplotype. MLC tests and HLA-D typing revealed that the homozygous C2-deficient patient is also homozygous at theHLA-D locus for the w2 specificity. Evidence was obtained for a heterogeneity of the HLA-Dw2 specificity. This observation confirms the remarkable association between C2 deficiency and theHLA-A10,B18,Dw2 haplotype.     

Phage typing ofSalmonella Typhi in the Netherlands

Summary A survey has been given of the results of phage typing of strains ofS. typhi found in Holland. It has been shown, that type A includes a different group of strains in systems drawn up with different Vi phages. An auxiliary system — to be used besides the system ofCraigie andYen — and a few new types, have been described.     

Human allospecific TLCs generated against HLA antigens associated with DRl through DRw8

We have studied the complexity and fine specificity of the HLA-D region using a panel of T lymphocyte clones generated against alloantigens associated with HLA-DR1 through DRw8. After extensive testing in population studies, 89 clones were tested in proliferation assays with 14 families. Segregation patterns were analyzed for haplotype associations by calculating sequential lod scores to test the likelihood that genes encoding epitopes detected by TLCs were linked to HLA genes. Four general categories were identified: (1) clonal responses that segregated with the same HLA-D region haplotype in all informative pedigrees; (2) clonal responses that segregated with HLA in all pedigrees but not always with the same haplotype; (3) clonal responses that segregated with HLA in some families but failed to segregate in others or produced equivocal results; (4) clonal responses that did not segregate with HLA haplotypes.Abbreviations used in this paper cpm counts per minute - DNV double normalized value - EBV Epstein-Barr virus - FCS fetal calf serum - HLA human MHC - HTC homozygous typing cell - LCL lymphoblastoid cell line - MHC major histocompatibility complex - MLC mixed lymphocyte culture - mAb monoclonal antibody - PBL peripheral blood lymphocyte - PLT primed lymphocyte typing - T-max maximized T test analysis - TCGF T-cell growth factor - TLC T-lymphocyte clone     

Complotype genetic loci segregate more frequently with HLA-DR than with HLA-B

The loci for BF, C2, C4A, and C4B are very closely linked to each other so that alleles of these plasma protein markers occur in populations in linkage disequilibrium and are inherited as single genetic units called complotypes. These complotypes are coded by a DNA region of the short arm of chromosome 6 embracing approximately 100 kilobases, which serve as a marker of the major histocompatibility complex. We have studied the complotypes of nine families with known HLA-B/DR crossovers. In seven families, the complotypes were inherited with HLA-DR, including in one family with a double recombination. The haplotype HLA-A28, Cw1, B27, FC3, 20, DR4 of JTr resulted from two recombinations between HLA-A2, Cwl, B27, SC42, DR7 and HLA-A28, Cwx or Cw1, B37, FC3, 20, DR4. In the remaining two families (Ro and Lo) the complotypes were inherited with HLA-B. The haplotype A2, Cw5, Bw44, SC30, DR3 of StLo resulted from paternal recombination between the haplotypes A2, Cw5, Bw44, SC30, DR4 and A24, B8, SC01, DR3, and the haplotype A24, Cw4, Bw35, SC31, DR3 of NaRo resulted from maternal recombination between A24, Cw4, Bw35, SC31, DR4 and A26, Bw41, FC31, DR3. Our data suggest that the complotype region maps closer to HLA-D than to HLA-B.     

Recombination During In Vitro Evolution

Recombination, the swapping of large portions of genetic information between and among parental genotypes, can be applied to in vitro evolution experiments on functional nucleic acids. Both homologous and heterologous recombination can be achieved using standard laboratory techniques. In many cases, recombination can allow for the discovery of a ribozyme or DNAzyme phenotype that would not likely be encountered by reliance on point mutations alone. In addition, recombination can often aid in the discovery of global optima in sequence space and/or lessen the number of generations it would take to reach optima. Recombination is most efficiently used in combination with point mutations and applied after the first couple of rounds of selection but before high-fitness genotypes dominate the selection. The “recombination zone” describes that region of sequence space—defined by the residues that will ultimately participate in the function of the winning nucleic acid(s)—where recombination is expected to be the most beneficial in the search for high-fitness genotypes.[Reviewing Editor: Martin Kreitman]Author order determined by a single Bernoulli trial as implemented by RPS.     

Molecular typing of Salmonella enterica serovar Sofia in Australia by pulsed‐field gel electrophoresis and repetitive element PCR typing

Aims: In this study, we used two molecular fingerprinting methods to investigate the genetic and clonal relationship shared by Australian Salmonella Sofia isolates. Methods and Results: A total of 84 Australian Salm. Sofia isolates from various states in Australia were typed using pulsed‐field gel electrophoresis (PFGE) (XbaI and SpeI) and repetitive element PCR (REP1R‐I primer). The previous problem of DNA degradation of Salm. Sofia strains was solved by modifying the lysis solution used to treat the bacterial plugs, allowing Salm. Sofia to be subtyped using PFGE. Molecular typing of isolates resulted in the generation of eight XbaI, six SpeI and five REP1 pattern profiles. Individual typing methods showed low discrimination index values (<0·5), indicating the poor discriminatory ability of the methods. However, the combination of the typing methods was able to improve the discrimination of isolates, further dividing them into 16 subtypes and raising the index value to 0·721. Conclusions: The combination of typing methods was shown to be the best approach to fingerprint Salm. Sofia. The Australian Salm. Sofia isolates only showed limited genetic diversity and probably share a clonal relationship. A majority of the Salm. Sofia isolates were not geographically restricted with the predominant pattern subtype observed amongst the isolates from various states. Significance and Impact of the Study: We have successfully devised a PFGE protocol that counteracts DNase activity of Salm. Sofia, enabling typing of this serovar.     

Large‐scale generation of megakaryocytes from human embryonic stem cells using transgene‐free and stepwise defined suspension culture conditions

ObjectivesEx vivo engineered production of megakaryocytes (MKs) and platelets (PLTs) from human pluripotent stem cells is an alternative approach to solve shortage of donor‐donated PLTs in clinics and to provide induced PLTs for transfusion. However, low production yields are observed and the generation of clinically applicable MKs and PLTs from human pluripotent stem cells without genetic modifications still needs to be improved.Materials and MethodsWe defined an optimal, stepwise and completely xeno‐free culture protocol for the generation of MKs from human embryonic stem cells (hESCs). To generate MKs from hESCs on a large scale, we improved the monolayer induction manner to define three‐dimensional (3D) and sphere‐like differentiation systems for MKs by using a special polystyrene CellSTACK culture chamber.ResultsThe 3D manufacturing system could efficiently generate large numbers of MKs from hESCs within 16‐18 days of continuous culturing. Each CellSTACK culture chamber could collect on an average 3.4 × 10⁸ CD41⁺ MKs after a three‐stage orderly induction process. MKs obtained from hESCs via 3D induction showed significant secretion of IL‐8, thrombospondin‐1 and MMP9. The induced cells derived from hESCs in our culture system were shown to have the characteristics of MKs as well as the function to form proPLTs and release PLTs. Furthermore, we generated clinically applicable MKs from clinical‐grade hESC lines and confirmed the biosafety of these cells.ConclusionsWe developed a simple, stepwise, 3D and completely xeno‐free/feeder‐free/transgene‐free induction system for the generation of MKs from hESCs. hESC‐derived MKs were shown to have typical MK characteristics and PLT formation ability. This study further enhances the clinical applications of MKs or PLTs derived from pluripotent stem cells.     

Microsatellite typing of the rhesus macaque MHC region

To improve the results gained by serotyping rhesus macaque major histocompatibility complex (MHC) antigens, molecular typing techniques have been established for class I and II genes. Like the rhesus macaque Mamu-DRB loci, the Mamu-A and -B are not only polymorphic but also polygenic. As a consequence, sequence-based typing of these genes is time-consuming. Therefore, eight MHC-linked microsatellites, or short tandem repeats (STRs), were evaluated for their use in haplotype characterization. Polymorphism analyses in rhesus macaques of Indian and Chinese origin showed high STR allelic diversity in both populations but different patterns of allele frequency distribution between the groups. Pedigree data for class I and II loci and the eight STRs allowed us to determine extended MHC haplotypes in rhesus macaque breeding groups. STR sequencing and comparisons with the complete rhesus macaque MHC genomic map allowed the exact positioning of the markers. Strong linkage disequilibria were observed between Mamu-DR and -DQ loci and adjacent STRs. Microsatellite typing provides an efficient, robust, and quick method of genotyping and deriving MHC haplotypes for rhesus macaques regardless of their geographical origin. The incorporation of MHC-linked STRs into routine genetic tests will contribute to efforts to improve the genetic characterization of the rhesus macaque for biomedical research and can provide comparative information about the evolution of the MHC region.     

A cytogenetic map on the entire length of rye chromosome 1R,including one translocation breakpoint,three isozyme loci and four C-bands

Summary A cytogenetic map of the whole 1 R chromosome of rye has been made, with distances between adjacent markers shorter than 50% recombination. Included in the map are isozyme loci Gpi-R1, Mdh-R1 and Pgd2, the telomere C-bands of the short arm (ts1) and the long arm (tl1), two interstitial C-bands in the short arm proximal to the nuclear organizing region (NOR) (is1) and in the middle of the long arm (il1), respectively, and translocation T273W (Wageningen tester set). By means of electron microscope analysis of spread pachytene synaptonemal complexes, the breakpoint of this translocation was physically mapped in the short arm of 1R, proximal to NOR, and in the long arm of 5R (contrary to previous assumptions). The data indicated the marker order: ts1 — Gpi-R1 — is1 — T273W/Mdh-R1 — il1 — Pgd2 — tl1. A comparison between genetic and physical maps revealed that recombination is mainly restricted to the distal regions of both arms. For the translocation T273W, in heterozygotes no recombinants were observed between the translocation breakpoint and its two adjacently located markers (is1 and Mdh-R1), but recombination was not reduced in the distal regions of the chromosome. The segregations of several other isozyme and C-band markers also analyzed in the investigation presented here were consistent with observations of earlier authors concerning chromosome asignment and linkage relationships.     

A recombination hotspot in the maize A1 intragenic region

Summary We find that recombination between two alleles of the maize A1 locus that contain transposon insertions at known molecular positions can occur at 0.04–0.08 cM per kbp (centimorgan per kilobase pair), which is two orders of magnitude higher than the recombination rate for the whole maize genome. It is however, close to the rates found within the bronze locus, another maize structural gene for which both genetic and molecular data are available. This observation supports the idea that the genome consists of regions that are highly recombinogenic — in some cases, at least, structural genes — interspersed with regions that are less recombinogenic.     

SSV1-encoded site-specific recombination system in Sulfolobus shibatae

Summary We present evidence for the existence of a conservative site-specific recombination system in Archaea by demonstrating integrative recombination of Sulfolobus shibatae virus SSV1 DNA with the host chromosome, catalysed by the SSVI-encoded integrase in vitro. The putative int gene of SSV1 was expressed in Escherichia coli yielding a protein of about 39 kDa. This protein alone efficiently recombined linear DNA substrates containing chromosomal (attA) and viral (attP) attachment sites; recombination with either negatively or positively supercoiled SSV1 DNA was less efficient. Intermolecular attA × attA and attP × attP recombination was also promoted by the SSV integrase. The invariant 44 by common attachment core present in all att sites contained sufficient information to allow recombination, whilst the flanking sequences effected the efficiency. These features clearly distinguish the SSV1 — encoded site — specific recombination system from others and make it suitable for the study of regulatory mechanisms of SSV1 genome — host chromosome interaction and investigations of the evolution of the recombination machinery.     

Linkage group IV of fish of the genus Xiphophorus (Poeciliidae): Assignment of loci coding for pyruvate kinase-1, glucosephosphate isomerase-1, and isocitrate dehydrogenase-1

Electrophoretic variation ascribable to three enzyme loci, coding for a pyruvate kinase (PK1), a glucose phosphate isomerase (GPI1), and an isocitrate dehydrogenase (IDH1), was observed in three species of fish of the genus Xiphophorus. Electrophoretic patterns in F₁ hybrid heterozygotes confirmed the dimeric structures of GPI and IDH, and indicated a multimeric structure for pyruvate kinase. Variant alleles at the three loci exhibited normal Mendelian segregation in backcross hybrids. Linkage analyses indicate a gene order and estimated recombination of PK1—10%—GPI1—41%—IDH1. No significant interference or sex- or population-specific recombination difference was detected. This group (designated linkage group IV) was shown to assort independently from the nine loci comprising linkage groups I, II, and III and from 23 other informative markers, within the limits of the data. No conclusions with respect to homology of linkage relationships could be reached, due to the presence of presumably duplicated loci in these fish coding for isozymes whose homology with enzymes in other vertebrate species is as yet unestablished.This work was supported in part by Public Health Service Research Grant CA-28909.     

Electrophoretic variation in human serum ceruloplasmin: A new genetic polymorphism

Through the application of a specific oxidase stain to results of starch gel electrophoresis of human serum, three different electrophoretic forms of ceruloplasmin—denoted CpA (fast), CpB (intermediate), and CpC (slow)—have been defined. The electrophoretic differences are small and were first recognized through a rare variant individual who had only the fast and slow forms. Five phenotypes displaying different combinations of the three electrophoretic forms have been defined in American Negroes; these are called CpA, CpAB, CpB, CpAC, and CpBC. Twin, family, and population studies have yielded evidence indicating that the A and B electrophoretic forms are controlled by a pair of autosomal codominant alleles, designated Cp ^A and Cp ^B , and suggesting that the C form may be determined by a third allele, Cp ^C , at the same locus. The variants constitute a genetic polymorphism in American Negroes, but occur only rarely in Caucasians.Supported by U.S. Atomic Energy Commission Contract AT(11-1)-1552, by U.S. Public Health Service Research Grants AM 09381 and HD 02083, and by U.S. Public Health Service Career Development Awards 6-K3-HE-24, 980 (DCS) and 1-K3-A-7959 (GJB).     

Population-specific recombination sites within the human MHC region

Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European and African were used to generate phased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from the phased haplotype data revealed discreet EHH drops corresponding to recombination events and these signatures were observed to be different for each population. Surprisingly, the majority of recombination sites detected are unique to each population, rather than being common. Unique recombination sites account for 56.8% (21/37 of total sites) in the Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38 sites) in Africans. Validation carried out at a known sperm typing recombination site of 45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrow the recombination region to 826 bp, and this was further refined to 660 bp by resequencing. This approach significantly enhanced mapping of the genomic architecture within the human MHC, and will be useful in studies to identify disease risk genes.     

Telomeric satellite DNA functions in regulating recombination

Molecular and cytogenetical analyses of three sibling species of Australian grasshopper, Atractomorpha australis, A. species-1 and A. similis, resolves one of the long standing problems of highly repeated DNA. In this system satellite DNA functions in regulating the level and position of recombination, irrespective of whether the repeated DNA is located in telomeric or centric regions. — Even though the three species do not differ in their euchromatic genome sizes, their relative DNA contents are 1.00/1.10/ 1.41, the difference in genome size being due solely to visible centric or telomeric blocks of heterochromatin. — Antibiotic analytical and preparative ultracentrifugation, in situ hybridization and renaturation kinetic analyses reveal that a large cryptic satellite of A. similis constitutes the heterochromatic telomeric blocks of nearly all autosomes and that the DNA of this satellite is highly repeated. — Comparison of these grasshopper data with published literature of heterochromatic rearrangements in Drosophila and with heterochromatin distribution and recombination patterns in diploid plant species reveals that in every case heterochromatin is implicated in some form of alteration in the meiotic recombination system.     

Immunogenetic polymorphism of lipoproteins in swine

Five new antigenic markers (allotypes) of swine serum lipoproteins are described. Specific antiallotype reagents were obtained from alloimmune precipitating sera. Identification studies and genetic analysis indicate that the five serum alloantigens—designated Lpp6, Lpp11, Lpp12, Lpp13, and Lpp14—are markers of low-density lipoproteins (LDL),d 1.002–1.075 g/ml, and are members of a previously described Lpp system. The Lpp6 allotype belongs to the group of individual markers and is determined by a new codominant allelic gene,Lpp ⁶, whereas the remaining four antigens—Lpp11, Lpp12, Lpp13, and Lpp 14—named common specificities, behave as alternative variants to Lpp1, Lpp2, Lpp3, and Lpp4, respectively, forming pairs of mutually exclusive alloantigens. Each Lpp gene in a heterozygous animal expresses itself independently on separate molecules and each haplotype carries one individual and at least four common specificities. The relationship between common and individual specificities, together with their number in the complex haplotypes, seems to shed some light on evolution of Lpp genes. It is proposed in this concept that the original gene for low-density lipoproteins in swine, and also in rhesus monkeys and the human, consisted of genetic information for common specificities only, the individual specificities evolving later as a result of point mutations.     

Linkage of lactate dehydrogenase-2, Ldh-2, in the mouse

An electrophoretically detectable variant of lactate dehydrogenase-2 in Mus musculus has been found and used to locate the structural gene, Ldh-2, on chromosome 6. Gene order and recombination frequencies are estimated as Sig—36.0±4.8—Lc 21.0±4.1—Mi ^wh—20.0±4.0—Ldh-2.