Adipose invasion of muscle in Wagyu cattle: Monitoring by histology and melting temperature.

Remarkably, Wagyu cattle progressively desaturate intramuscular and subcutaneous fat leading to melting temperatures (Tm) well below 38°C. In parallel, the adipose tissue expands, arborises and invades the muscle. The process is aggressive in that it leads to loss of myofibres resulting in much smaller fascicles and therefore fine marbling or snowflaking. The "Microscopic score" appears to be an excellent measure of marbling especially for lesser and greater degrees which are not quantified reliably by others methods. By comparing muscle groups, we conclude that the tailhead is a suitable site for sequential monitoring. Melting temperatures of intramuscular and subcutaneous tissue are also useful.

A novel method of measuring the melting point of animal fats.

The melting point (TM) of fat is relevant to health, but available methods of determining TM are cumbersome. One of the standard methods of measuring TM for animal and vegetable fats is the slip point, also known as the open capillary method. This method is imprecise and not amenable to automation or mass testing. We have developed a technique for measuring TM of animal fat using the Rotor-Gene Q (Qiagen, Hilden, Germany). The assay has an intra-assay SD of 0.08°C. A single operator can extract and assay up to 250 samples of animal fat in 24 h, including the time to extract the fat from the adipose tissue. This technique will improve the quality of research into genetic and environmental contributions to fat composition of meat.

Evaluation of the class II region of the major histocompatibility complex of the greyhound with the genomic matching technique and sequence-based typing.

The class II region of the major histocompatibility complex was evaluated in 25 greyhounds by sequence-based typing and the genomic matching technique (GMT). Two new DLA-DRB1 alleles were identified. Twenty-four dogs carried the DLA-DRB1*01201/DQA1*00401/DQB1*01303/DQB1*01701 haplotype, which carries two DQB1 alleles. One haplotype was identified from which DQB1 and DQA1 appeared to be deleted. The GMT enabled detection of DQB1 copy number, discrimination of the different class II haplotypes and the identification of new, possibly biologically relevant polymorphisms.

Epistasis between the MHC and the RCA alpha block in primary Sjögren syndrome.

OBJECTIVE: The RCA alpha block (Regulators of Complement Activation, 1q32) contains critical complement regulatory genes such as CR1 and MCP. This study examined RCA alpha block haplotype associations with both disease susceptibility and diversification of the anti-Ro/La autoantibody response in primary Sjögren syndrome (pSS). METHODS: 115 patients with pSS and 98 controls were included in the study. 93 of 109 (85%) of the patients with pSS were seropositive for Ro/La autoantibodies. The Genomic Matching Technique (GMT) was used to define RCA alpha block ancestral haplotypes (AH). RESULTS: RCA alpha block haplotypes, AH1 and AH3, were both associated with autoantibody-positive pSS (p = 0.0003). Autoantibody associations with both HLA DR3 and DR15 have been previously defined. There was an epistatic interaction (p = 0.023) between RCA alpha AH1 and HLA DR3, and this genotypic combination was present in 48% of autoantibody-positive patients with pSS compared with 8% of controls. This epistasis is most simply attributable to an interaction between C4 and its receptor, CR1, encoded within the RCA alpha block. Both DR3 and a relative C4 deficiency are carried on the major histocompatibility complex 8.1 ancestral haplotype. Only four of 92 (4%) autoantibody-positive patients with pSS did not carry any risk RCA alpha or HLA haplotype, compared with 36 of 96 (38%) controls, and there were differences in haplotype frequencies within autoantibody subsets of pSS. CONCLUSIONS: Normal population variation in the RCA alpha block, in addition to the major histocompatibility complex, contributes genetic susceptibility to systemic autoimmune disease and the autoantibody response. This finding provides evidence for the role of regulation of complement activation in disease pathogenesis.

Haplotyping of the canine MHC without the need for DLA typing.

The genomic matching technique has proven useful in MHC haplotyping in humans. We have adopted a similar approach in Australian cattle dogs and report that genotyping can be achieved with a single assay.

Use of the genomic matching technique to complement multiplex STR profiling reduces DNA profiling costs in high volume crimes and intelligence led screens.

The genomic matching technique (GMT) targets duplicated polymorphic sequences within genomic blocks in the human major histocompatibility complex (MHC), differentiating between individuals at the DNA level using a single primer pair per block. The GMT is currently used to supplement human leukocyte antigen (HLA) typing to match donor and recipient pairs for bone marrow transplantation and has the potential to be employed as a powerful exclusion tool in forensic biology. The GMT is highly reproducible, produces DNA profiles from less than 1 ng of DNA and was successfully employed to profile a range of forensic samples including buccal swabs, handled objects and fingerprints. Furthermore, GMT profiles from a single genomic block in the MHC are likely to be more discriminatory than known highly polymorphic short tandem repeat (STR) loci such as ACTBP2. As such, the GMT can reduce the cost of investigations that require profiling of multiple suspects or samples from one or more crime scenes and could be extended to profile genomic blocks in other polymorphic genetic systems in the human genome.

Indels and imperfect duplication have driven the evolution of human Complement Receptor 1 (CR1) and CR1-like from their precursor CR1 alpha: importance of functional sets.

This study examines the effects of duplication and insertions-deletions (indels) by comparing human complement receptor 1 (CR1) and human CR1-like (CR1L) with syntenic genes from four other vertebrates (chimpanzee, baboon, rat, and mouse). By phylogenetic analysis, the domains of these genes can be classified into 10 distinct subfamilies (a, b, c, d, e, f, g(-like), h, j, and k), which have been largely conserved throughout vertebrate and invertebrate evolution. In spite of many complex and diverse duplications and indels, the subfamily order of domains (a, j, e, f, b, k, d, g(-like)) has been maintained. The number of domain sets has increased progressively, thereby expanding the functional repertoire.

Genomic analysis reveals a duplication of eight rather than seven short consensus repeats in primate CR1 and CR1L: evidence for an additional set shared between CR1 and CR2.

We report the discovery of previously unrecognised short consensus repeats (SCRs) within human and chimpanzee CR1 and CR1L. Analysis of available genomic, protein and expression databases suggests that these are actually genomic remnants of SCRs previously reported in other complement control proteins (CCPs). Comparison with the nucleotide motifs of the 11 defined subfamilies of SCRs justifies the designation g-like because of the close similarity to the g subfamily found in CR2 and MCP. To date, we have identified five such SCRs in human and chimpanzee CR1, one in human and chimpanzee CR1L, but none in either rat or mouse Crry in keeping with the number of internal duplications of the long homologous repeat (LHR) found in CR1 and CR1L. In fact, at the genomic level, the ancestral LHR must have contained eight SCRs rather than seven as previously thought. Since g-like SCRs are found immediately downstream of d SCRs, we suggest that there must have been a functional dg set which has been retained by CR2 and MCP but which is degenerate in CR1 or CR1L. Interestingly, dg is also present in the CR2 component of mouse CR1. The degeneration of the g SCR must have occurred prior to the formation of primate CR1L and prior to the duplication events which resulted in primate CR1. In this context, the apparent conservation of g-like SCRs may be surprising and may suggest the existence of mechanisms unrelated to protein coding. These results provide examples of the many processes which have contributed to the evolution of the extensive repertoire of CCPs.

Genetics of human complement component C4 and evolution the central MHC.

The two classes of human complement component C4 proteins C4A and C4B manifest differential chemical reactivities and binding affinities towards target surfaces and complement receptor CR1. There are multiple, polymorphic allotypes of C4A and C4B proteins. A complex multiplication pattern of C4A and C4B genes with variations in gene size, gene dosage and flanking genes exists in the population. This is probably driven by the selection pressure to respond to a great variety of parasites efficiently and effectively, which the bony fish achieved through the multiplication and diversification of the related complement C3 proteins. Complement C4, C3 and C5 belong to the alpha2 macroglobulin protein family but acquired specific features that include an anaphylatoxin domain, a netrin (NTR) domain, and stretches of basic residues for proteolytic processings to form multiple chain structures. Complement C3 and C4 are important in the innate immune response as they opsonize parasites for phagocytosis. The emergence of complement C3 predates proteins involved in the adaptive immune response as C3 is present in deuterostome invertebrates such as echinoderms. The human C4 genes are located in the central MHC at chromosome 6p21.3. C3 and C5 are located at chromosome 19 and 9, respectively, with representatives of the other groups of genes paralogous to the MHC at 19p13.1-p13.3, 1q21-25, and 9q33-34. The central MHC also contains genes for complement components C2 and Bf. These genes appear to have similar evolutionary histories to C3/C4/C5 and are used here to illustrate stepwise processes resulting in co-location of diverse domains, chromosomal duplication, local segmental duplication and divergence of sequence and function. This model of evolution is useful in the investigation of innate and acquired immunity and in seeking explanations for diseases associated with MHC ancestral haplotypes.

The association between HLA-A alleles and an Alu dimorphism near HLA-G.

The AluYb8 sequences are a subfamily of short interspersed Alu retroelements that have been amplified within the human genome during recent evolutionary time and are useful polymorphic markers for studies on the origin of human populations. We have identified a new member of the Yb8 subfamily, AluyHG, located between the HLA-H and -G genes and 88-kb telomeric of the highly polymorphic HLA-A gene within the alpha block of the major histocompatibility complex (MHC). The AluyHG element was characterised with a view to examining the association between AluyHG and HLA-A polymorphism and reconstructing the history of the MHC alpha block. A specific primer pair was designed for a simple PCR assay to detect the absence or presence (dimorphism) of the AluyHG element within the DNA samples prepared from a panel of 46 homozygous cell-lines containing complete or recombinant ancestral haplotypes (AH) of diverse ethnic origin and 92 Caucasoid and Asian subjects on which HLA-A typing was available. The AluyHG insertion was most strongly associated with HLA-A2 and, to a lesser degree with HLA-A1, -A3, -A11, and A-19. The gene frequency of the AluyHG insertion for 146 Caucasians and 94 Chinese-Han was 0.30 and 0.32 and there was no significant difference between the observed and expected frequencies. The results of the association studies and the phylogenetic analysis of HLA-A alleles suggest that the AluyHG sequence was integrated within the progenitor of HLA-A2, but has been transferred by recombination to other human ancestral populations. In this regard, the dimorphic AluyHG element is an important diagnostic marker for HLA association studies and could help in elucidating the evolution and functions of the MHC alpha block and polymorphism within and between ancestral haplotypes.

Sequence analysis of the MHC class I region reveals the basis of the genomic matching technique.

The genomic matching technique (GMT) improves survival following bone marrow transplantation (BMT) between unrelated donor and recipient pairs correlating with a decrease in incidence and severity of graft-versus-host disease (GvHD). The principles of this technique are based on the duplication and polymorphic characteristics of the major histocompatibility complex (MHC). Specifically, the beta block GMT matches for a 300 kb region that contains the human leukocyte antigen (HLA-B and -C) genes as well as other non-HLA genes such as the natural killer cell receptor ligand PERB11 (MIC). The block contains two large segmental duplications. One results in two PERB11 genes (11.1 and 11.2), the other in two class I genes (HLA-B and -C). With the complete sequencing of the class I region of the MHC in different haplotypes, we can now show that the beta block GMT profiles reflect amplification of the duplicated PERB11 segments and not the duplicated segments containing HLA-B and -C, and yet provide a signature that characterizes the entire block rather than individual loci.

What is science good for?

A nonbusiness discipline can provide a useful framework for thinking about old problems in new ways. People who study management, for instance, freely borrow from many fields of science to theorize about organizational behavior and business strategy. Evolutionary psychology and biology are especially popular sources of inspiration. But should they be? Evolutionary biologist Richard Dawkins has spent much of his career explaining science to the public. More than 20 years ago, his book The Selfish Gene shattered the popular belief that evolution necessarily favors altruism and self-sacrifice. In a conversation with HBR senior editor Diane Coutu, Dawkins discusses the role of science in our lives and identifies some of the more glaring public misperceptions of scientific theories. In particular, he disentangles the current notion that certain behaviors are in some way preprogrammed and explodes some contemporary myths about the Human Genome Project. Dawkins says much of the popular fear surrounding genetic manipulation is unfounded. "Humans have been practicing it for thousands of years, to no obvious ill effect," he says. Modern foot-long corncobs, the result of more than 1,000 years of artificial selection, are "quite Frankenstein-like" compared to their half-inch-long progenitors, he points out. He also touches on agriculture giant Monsanto and the media: "Part of the reason for Monsanto's troubles is that the company came up against an extraordinary amount of unfortunate, even malevolent, media hype," he says. "And people were more or less misled, by one scare story after another, into stampeding." A staunch defender of science as a haven of rational thought, Dawkins counsels businesspeople to recognize the limitations--as well as the beauty--of science.

SNP profile within the human major histocompatibility complex reveals an extreme and interrupted level of nucleotide diversity.

The human major histocompatibility complex (MHC) is characterized by polymorphic multicopy gene families, such as HLA and MIC (PERB11); duplications; insertions and deletions (indels); and uneven rates of recombination. Polymorphisms at the antigen recognition sites of the HLA class I and II genes and at associated neutral sites have been attributed to balancing selection and a hitchhiking effect, respectively. We, and others, have previously shown that nucleotide diversity between MHC haplotypes at non-HLA sites is unusually high (>10%) and up to several times greater than elsewhere in the genome (0.08%-0.2%). We report here the most extensive analysis of nucleotide diversity within a continuous sequence in the genome. We constructed a single nucleotide polymorphism (SNP) profile that reveals a pattern of extreme but interrupted levels of nucleotide diversity by comparing a continuous sequence within haplotypes in three genomic subregions of the MHC. A comparison of several haplotypes within one of the genomic subregions containing the HLA-B and -C loci suggests that positive selection is operating over the whole subgenomic region, including HLA and non-HLA genes. [The sequence data for the multiple haplotype comparisons within the class I region have been submitted to DDBJ/EMBL/GenBank under accession nos. AF029061, AF029062, and AB031005-AB031010. Additional sequence data have been submitted to the DDBJ data library under accession nos. AB031005-AB03101 and AF029061-AF029062.]

The implications of intergenic polymorphism for major histocompatibility complex evolution.

A systematic survey of six intergenic regions flanking the human HLA-B locus in eight haplotypes reveals the regions to be up to 20 times more polymorphic than the reported average degree of human neutral polymorphism. Furthermore, the extent of polymorphism is directly related to the proximity to the HLA-B locus. Apparently linkage to HLA-B locus alleles, which are under balancing selection, maintains the neutral polymorphism of adjacent regions. For these linked polymorphisms to persist, recombination in the 200-kb interval from HLA-B to TNF must occur at a low frequency. The high degree of polymorphism found distal to HLA-B suggests that recombination is uncommon on both sides of the HLA-B locus. The least-squares estimate is 0.15% per megabase with an estimated range from 0.02 to 0.54%. These findings place strong restrictions on possible recombinational mechanisms for the generation of diversity at the HLA-B.

Further characterization of MHC haplotypes demonstrates conservation telomeric of HLA-A: update of the 4AOH and 10IHW cell panels.

Cell panels have been used extensively in studies of polymorphism and disease associations within the major histocompatibility complex (MHC), but the results from these panels require continuous updates with the increasing availability of novel data. We present here an updated table of the typings of the 10IHW and 4AOH panels. Local data included are HFE, HERV-K(C4) and six microsatellites telomeric of HLA-A. Typings for class I, MICA (PERB11.1), MICB (PERB11.2), XA, XB, LMP2 and 10 microsatellites reported by others have also been consolidated in this table. The tabulation shows that the length of conservation in the human MHC is even more extensive than previously thought. Human MHC ancestral haplotypes are inherited as a conserved region of genomic sequence spanning some 6-8 megabases from the HLA class II region and beyond the HLA class I region up to and including the HFE gene. Numerous examples of historical recombinations were also observed.

Using alu J elements as molecular clocks to trace the evolutionary relationships between duplicated HLA class I genomic segments.

The class I region of the major histocompatibility complex contains two subgenomic blocks (250-350 kb each), known as the alpha and beta blocks. These blocks contain members of multicopy gene families including HLA class I, HERV-16 (previously called P5 sequences), and PERB11 (MIC). We have previously shown that each block consists of imperfect duplicated segments (duplicons) containing linked members of different gene families, retroelements and transposons that have coevolved as part of two separate evolutionary events. Another region provisionally designated here as the kappa block is located between the alpha and the beta blocks and contains HLA-E, -30, and -92, HERV-16 (P5.3), and PERB11.3 (MICC) within about 250 kb of sequence. Using Alu elements to trace the evolutionary relationships between different class I duplicons, we have found that (a) the kappa block contains paralogous (duplicated) Alu J sequences and other retroelement patterns more in common with the beta than the alpha block; (b) the retroelement pattern associated with the HLA-E duplicon is different from all other HLA class I duplicons, indicating a more complex evolution; (c) the HLA-92 duplicon, although substantially shorter, is closely related in sequence to the HLA-B and -C duplicons; (d) two of the six paralogous Alu J elements within the HLA-B and -C duplicons are associated with the HLA-X duplicon, confirming their evolutionary relationships within the beta block; and (e) the paralogous Alu J elements within the alpha block are distinctly different from those identified within the beta and kappa blocks. The sequence conservation and location of duplicated (paralogous) Alu J elements in the MHC class I region show that the beta and kappa blocks have evolved separately from the alpha block beginning at a time before or during the evolution of Alu J elements in primates.

Coevolution of HLA-B and PERB11.1 (MICA): significance of independent triplet expansion within the transmembrane region of PERB11.1 (MICA).

Several highly polymorphic sequences are present in the beta block of the MHC, especially HLA-B, HLA-C, PERB11.1 (MICA), and PERB11.2 (MICB). It is now apparent that the polymorphism of PERB11.1 is of the same order as that of HLA-A, -B, and -C and it has been suggested that PERB11 could explain some of the disease associations previously attributed to HLA-B. Phylogenetic analysis of PERB11 alpha-domain sequences demonstrates relationships with HLA-B cross-reactive serogroups. In contrast, the transmembrane polymorphisms do not appear to be associated with either PERB11 or HLA-B. These data indicate that PERB11 and HLA-B have evolved in concert from their common ancestors and that the transmembrane polymorphisms have arisen independently and more recently. MHC disease associations will need to be reviewed in the light of mechanisms such as receptor binding and signaling.

Duplication and diversification of the apolipoprotein CI (APOCI) genomic segment in association with retroelements.

We have previously shown that several multicopy gene families within the major histocompatibility complex (MHC) arose from a process of segmental duplication. It has also been observed that retroelements play a role in generating diversity within these duplicated segments. The objective of this study was to compare the genomic organization of a gene duplication within another multicopy gene family outside the MHC. Using new continuous genomic sequence encompassing the APOE-CII gene cluster, we show that APOCI and its pseudogene, APOCI', are contained within large duplicated segments which include sequences from the hepatic control region (HCR). Flanking Alu sequences are observed at both ends of the duplicated unit, suggesting a possible role in the integration of these segments. As observed previously within the MHC, the major differences between the segments are the insertion of sequences (approximately 200-1000 bp in length), consisting predominantly of Alu sequences. Ancestral retroelements also contribute to the generation of sequence diversity between the segments, especially within the 3' poly(A) tract of Alu sequences. The exonic and regulatory sequences of the APOCI and HCR loci show limited sequence diversity, with exon 3 being an exception. Finally, the typing of pre- and postduplication Alus from both segments indicates an estimated time of duplication of approximately 37 million years ago (mya), some time prior to the separation of Old and New World monkeys.

PERB11 (MIC): a polymorphic MHC gene is expressed in skin and single nucleotide polymorphisms are associated with psoriasis.

The susceptibility genes for psoriasis remain to be identified. At least one of these must be in the major histocompatibility complex (MHC) to explain associations with alleles at human leucocyte antigen (HLA)-A, -B, -C, -DR, -DQ and C4. In fact, most of these alleles are components of just two ancestral haplotypes (AHs) designated 13.1 and 57.1. Although relevant MHC gene(s) could be within a region of at least 4 Mb, most studies have favoured the area near HLA-B and -C. This region contains a large number of non-HLA genes, many of which are duplicated and polymorphic. Members of one such gene family, PERB11.1 and PERB11.2, are expressed in the skin and are encoded in the region between tumour necrosis factor and HLA-B. To investigate the relationship of PERB11.1 alleles to psoriasis, sequence based typing was performed on 97 patients classified according to age of onset and family history. The frequency of the PERB11.1*06 allele is 44% in type I psoriasis but only 7% in controls (Pc = 0.003 by Fisher's exact test, two-tailed). The major determinant of this association is a single nucleotide polymorphism (SNP) within intron 4. In normal and affected skin, expression of PERB11 is mainly in the basal layer of the epidermis including ducts and follicles. PERB11 is also present in the upper keratin layers but there is relative deficiency in the intermediate layers. These findings suggest a possible role for PERB11 and other MHC genes in the pathogenesis of psoriasis.

Extensive nucleotide variability within a 370 kb sequence from the central region of the major histocompatibility complex.

The recent availability of the genomic sequence spanning the central and telomeric end of the major histocompatibility complex (MHC) has allowed a detailed study of its organisation, gene content and level of nucleotide variability. Previous analyses of nucleotide variability in the MHC have focused on the coding regions of the human leukocyte antigen (HLA) Class I and II genes. Non-coding nucleotide variability has been considered a by-product of exonic diversity. However, with the advent of genomic sequencing, the extent of non-coding nucleotide variability within the MHC has just begun to be appreciated. In this study, we compared different human haplotypes in 370 kb of sequence in the central region of the MHC to show the following: 1. unusually high levels of non-coding nucleotide variability, up to 80 times greater than elsewhere in the genome; 2. non-coding nucleotide variability greater than 1% at nucleotide sites distant to the Class I genes; 3. nucleotide variability greater than 1% maintained over regions containing highly linked loci; and 4. distinct troughs and peaks in the level of nucleotide variability. We will discuss these observations in relation to a possible role of nucleotide variability in the organisation of the MHC.