The pineal region in the opossum,Didelphis virginiana

Summary In the pineal region of the opossum, Didelphis virginiana, two types of cells predominate: 1) pinealocytes, and 2) fibrous astrocytes. Pinealocytes are characterized by the presence of prominent Golgi bodies, numerous clear and dense-cored vesicles, sensory cilia (9+0), vesicle-crowned rods, and condensation of a material that was always associated with the rough endoplasmic reticulum. In addition, two other cell types are occasionally seen. These include 1) neuron-like cells, and 2) darker staining cells of unknown identity. The endoplasmic reticulum of the darker staining cells is typically expanded and filled with an amorphous substance. Although the pineal region is small in size, the present findings suggest that pinealocytes in this species are metabolically active cells displaying a secretory function. Moreover, the presence of sensory cilia (9+0) and vesicle-crowned rods indicates that pinealocytes of the opossum are phylogenetically related to the photoreceptor cells found in the pineal organ of lower vertebrates.     

Evolution of the ovine MHC DQA region

Southern hybridisation was used to define an apparent gene duplication event at the ovine DQA2 locus. Approximately 500 sheep from five different breeds were genotyped at their DQA1 and DQA2 loci. A subset of these were selected for further characterisation. Southern hybridisation of TaqI digested DNA revealed no DQA1 region in some sheep. It was also noted in these DQA1 null animals the DQA2 specific probe hybridised to two bands. An EcoRV-RFLP designed to distinguish copy number confirmed this duplication of the DQA2 region. The results showed that the duplication was exclusively associated with the DQA1 null haplotype and occurred only in alleles DQA2-F, -G, -I and -J. Comparison with bovine MHC genes revealed that they also contained a DQA1 null haplotype and that this haplotype was associated with a putative DQA3 gene. The potential for an ovine DQA3 locus is discussed.     

Population-specific recombination sites within the human MHC region

Genetic rearrangement by recombination is one of the major driving forces for genomeevolution, and recombination is known to occur in non-random, discreet recombination siteswithin the genome. Mapping of recombination sites has proved to be difficult,particularly, in the human MHC region that is complicated by both population variation andhighly polymorphic HLA genes. To overcome these problems, HLA-typed individualsfrom three representative populations: Asian, European and African were used to generatephased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from thephased haplotype data revealed discreet EHH drops corresponding to recombination eventsand these signatures were observed to be different for each population. Surprisingly, themajority of recombination sites detected are unique to each population, rather than beingcommon. Unique recombination sites account for 56.8% (21/37 of total sites) inthe Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38sites) in Africans. Validation carried out at a known sperm typing recombination site of45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrowthe recombination region to 826 bp, and this was further refined to 660 bpby resequencing. This approach significantly enhanced mapping of the genomic architecturewithin the human MHC, and will be useful in studies to identify disease risk genes.     

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.     

The Drosophila subobscura Adh genomic region contains valuable evolutionary markers.

We have sequenced 4 kb of the genomic region comprising the Adh (Alcohol dehydrogenase) gene of Drosophila subobscura. In agreement with other species which belong to the same subgenus, two structural genes, Adh and Adh-dup, are contained in this region. The main features of these two genes of D. subobscura have been inferred from the sequence data and compared with the homologous region of D. ambigua and D. pseudoobscura. Drosophila subobscura Adh and Adh-dup differ from those of D. ambigua at a corrected estimation of 10.1% and 12.5%, respectively, while from those of D. pseudoobscura they differ by 9.5% and 8.1%, respectively. Our data suggest that Adh and Adh-dup are evolving independently, showing a species-specific pattern. Moreover, particular features of some regions of these genes make them valuable evolutionary hallmarks. For instance, replacement substitutions in the third exon of Adh may indicate the branching of the melanogaster-obscura groups, whereas replacement substitutions in the third exon of the Adh-dup could be used to assess speciation within the obscura group.     

Molecular characterization of the porcine MHC class I region

A porcine cosmid library was screened with a human MHC class I cDNA. Four positive clones were isolated and mapped with different restriction endonucleases. Altogether nine SLA class I genes were identified and their positions located within restriction maps. Sizes of class I homologous DNA sequences varied between 3600 and 5800bp. The distances between these regions ranged from 11900 to 22200bp.     

Genetic organization of the ovine MHC class II region

To study the class II genes of the major histocompatibility region of the sheep genome, human HLA class II genes corresponding to the known subregions in man (DR, DQ, DP, DO, and DZ) were used for Southern hybridization analysis of sheep DNA and to probe a sheep genomic library. Hybridizing bands were noted for all probes except DP. DQ and and DR appear to be present as multicopy genes, while DR-, DZ-, , and DO -like genes appear to be single copy. All bands detected with the DP probe were also detectable with other chain probes. From eight -bacteriophage clones of a sheep genomic library nine distinct class II genes were identified. These genes were characterized by differential hybridization analysis and restriction mapping. Two genes were DR -like, three DQ-like and four DQ -like. The extensive cross-hybridization observed with chain probes was not seen with chain probes. The results of this study suggest that the major histocompatibility complex class II region of the sheep has a similar genetic organization to that of man, with the provisional exception of the DP subregion.Abbreviation used in this paper OLA ovine major histocompatibility complex     

The extensor assembly in two species of opossum,Philander opossum and Didelphis marsupialis

In considering primate and hominoid phylogeny, the fundamental position assigned to opossums is explained partially by the characteristic morphology of their hands and feet. One of the main functional features of the human hand is the ability to make a stabilized arch of the finger. Because the extensor assembly plays a key role in establishing an arched finger, the extensor systems of the digits of both the hands and feet were studied in two species of opossum, Philander opossum and Didelphis marsupialis. In the foot, two extensor tendons join in each toe to form one tendinous plate, which inserts onto the base of the second phalanx. Lumbricals join this plate along the tibial side, and interosseus insertions are found, although a true interosseus wing is lacking. At the proximal interphalangeal level, a terminal tendon takes its origin from this tendinous plate. This terminal tendon is oval in cross-section and contains elastic structures. Oblique bands arise from this terminal tendon and run proximally along the proximal interphalangeal joint inserting onto the base of the first phalanx. There are elastic structures in the flexor tendon on the dorsal side near its site of insertion. In the hand, the main extensor tendons are arranged differently and the interossei contribute substantially to the extensor assembly. Otherwise, the extensor assembly of the hands and feet are quite similar. The function of the so-called paratendinous intravaginal flexors is discussed as are evolutionary aspects of the extensor assembly.     

Isolation and characterization of three class II MHC genomic clones from the chicken

A genomic library was constructed from sperm DNA from an individual of the inbred chicken line G-B2, MHC haplotype B6. The library was screened with a chicken class II probe (beta 2 exon specific) and three MHC class II beta chain genomic clones were isolated. The restriction maps of the three clones showed that each of the three clones was unique. The position of the beta chain sequence was located in each of the three genomic clones by Southern blot hybridization. Subclones containing the beta chain gene were produced from each of the genomic clones and the orientation of the leader peptide, beta 1, beta 2, transmembrane, and cytoplasmic exons was determined by Southern blot hybridization and nucleotide sequencing. The complete nucleotide sequence of two of the three subclones was determined. Comparison of the nucleotide and predicted amino acid sequences of the two subclones with other class II beta chain sequences showed that the B6 chicken beta chain genes are evolutionarily related to the class II beta chain genes from chickens of other MHC haplotypes, and to class II beta chain genes from other species. Analysis of Southern blots of B6 chicken DNA, as well as the isolation of the three beta chain genes, suggests that chickens of the B6 haplotype possess at least three MHC class II beta chain genes.     

Structure and expression of two porcine genomic clones encoding class I MHC antigens

Two nonallelic porcine class I MHC (SLA) genes have been isolated and characterized. Both genes are expressed in mouse L cells, directing the synthesis of class I SLA molecules that carry common monomorphic determinants but are serologically distinct. The corresponding DNA sequences have been determined. The organization of both of these genes is similar to that of other class I genes: a leader exon, three exons encoding extracellular domains, a transmembrane exon, and three intracytoplasmic exons. The two genes are highly homologous in both exon and intron segments, with average homologies of 88% and 80%, respectively. Nucleotide changes in exon 2 are clustered, whereas those in the other exons are dispersed throughout. Comparison of the swine DNA sequences with class I genes from other species reveals a generally high conservation of exons 2, 3, 4, and 6 with lower homology in the remaining protein-encoding domains. Introns are markedly less well conserved, although moderate homology is found between swine and human class I MHC genes in both introns and 3' flanking regions. Taken together with comparisons of the deduced protein sequences, these data indicate an order of swine greater than human greater than rabbit greater than mouse in the relationship of class I genes.     

Comparative genomic analysis of two avian (quail and chicken) MHC regions

We mapped two different quail Mhc haplotypes and sequenced one of them (haplotype A) for comparative genomic analysis with a previously sequenced haplotype of the chicken Mhc. The quail haplotype A spans 180 kb of genomic sequence, encoding a total of 41 genes compared with only 19 genes within the 92-kb chicken Mhc. Except for two gene families (B30 and tRNA), both species have the same basic set of gene family members that were previously described in the chicken "minimal essential" Mhc. The two Mhc regions have a similar overall organization but differ markedly in that the quail has an expanded number of duplicated genes with 7 class I, 10 class IIB, 4 NK, 6 lectin, and 8 B-G genes. Comparisons between the quail and chicken Mhc class I and class II gene sequences by phylogenetic analysis showed that they were more closely related within species than between species, suggesting that the quail Mhc genes were duplicated after the separation of these two species from their common ancestor. The proteins encoded by the NK and class I genes are known to interact as ligands and receptors, but unlike in the quail and the chicken, the genes encoding these proteins in mammals are found on different chromosomes. The finding of NK-like genes in the quail Mhc strongly suggests an evolutionary connection between the NK C-type lectin-like superfamily and the Mhc, providing support for future studies on the NK, lectin, class I, and class II interaction in birds.