Biomedical subjects
S J O'Brien
Publications and source records attributed to S J O'Brien.
Emergence of the keratinocyte growth factor multigene family during the great ape radiation.
The structural gene for human keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, consists of three coding exons and two introns typical of other fibroblast growth factor loci. A portion of the KGF gene, located on chromosome 15, is amplified to approximately 16 copies in the human genome, and these highly related copies (which consist of exon 2, exon 3, the intron between them, and a 3' noncoding segment of the KGF transcript) are dispersed to multiple human chromosomes. The KGF-like sequences are transcriptionally active, differentially regulated in various tissues, and composed of three distinct classes of coding sequences that are 5% divergent from each other and from the authentic KGF sequence. Multiple copies of KGF-like genes were also discovered in the genomic DNAs of chimpanzee and gorilla but were not found in lesser apes (gibbon), Old World monkeys (African green monkey and macaques), mice, or chickens. The pattern of evolutionary occurrence suggests that a primordial KGF gene was amplified and chromosomally dispersed subsequent to the divergence of orangutan from African apes but before the trichotomous divergence of human, chimpanzee, and gorilla 5-8 million years ago. The appearance of a transcriptionally active and chromosomally dispersed multigene KGF family may have implications in the evolution of the great apes and humans.
Cellular repopulation of deep-frozen meniscal autografts: an experimental study in the dog.
This study evaluated the cellular repopulation of deep-frozen meniscal autografts. Medial menisci of adult dogs were excised, deep-frozen in liquid nitrogen (-196 degrees C) for 10 min, and orthotopically reimplanted into the joint. Deep-freezing was found to effectively kill all the cells within the meniscus as determined by the absence of Na(2)35SO4 incorporation. Following orthotopic replacement within the knee joint, menisci were repopulated with cells that seemed to originate from the adjacent synovium. These cells migrated over the surface of the meniscus and began to invade the deeper layers of the tissue. However, even after 6 months, the central core of the meniscus remained acellular. While the new cells appeared to modulate into cells that are similar in appearance to meniscal fibrochondrocytes, the exact phenotypic expression of these newly differentiated cells has yet to be determined. Histological alterations, as manifested by a loss of normal orientation of the collagen architecture of the superficial layers of the meniscus; was evident at 6 months and suggests that a remodeling phenomenon may be associated with the cellular repopulation. While biomaterial studies have not been carried out on these specimens, the morphologic alterations observed in the collagen orientation would suggest a possible alteration in the material properties of the repopulated meniscus. The clinical implication of this study is that the structural remodeling associated with the cellular repopulation of deep-frozen meniscal allografts may make the transplanted meniscus more susceptible to injury.
Detecting single base substitutions as heteroduplex polymorphisms.
We have developed a sensitive technique for detecting single base substitutions in polymerase chain reaction (PCR) products from individuals heterozygous for polymorphisms or new mutations. This technique takes advantage of the formation of heteroduplexes in the PCR between different alleles from heterozygous individuals. These heteroduplexes can be detected on polyacrylamide gels because they migrate slower than their corresponding homoduplexes. Using PCR, we have generated a series of point mutations in a defined region of DNA in the equine infectious anemia virus (EIAV). Each mutation is the result of a single base substitution. By mixing the PCR products amplified from these mutations with one another, as well as with wildtype PCR products, we can generate heteroduplexes in which the identity of the mismatched bases is known. We detected eight of nine point mutations using this technique. We have also modified the electrophoretic conditions to optimize the detection of these heteroduplexes. In addition, the usefulness of this technique is demonstrated by its ability to detect a mutation in the cystic fibrosis gene that is the result of a single base substitution. This technique should prove useful for rapidly screening large numbers of individuals for new mutations or polymorphisms.
Feline arylsulfatase B (ARSB): isolation and expression of the cDNA, comparison with human ARSB, and gene localization to feline chromosome A1.
Arylsulfatase B (ARSB) is the lysosomal enzyme that catalyzes the hydrolysis of 4-sulfate groups from N-acetylgalactosamine 4-sulfate moieties on the glycosaminoglycans, dermatan sulfate and chondroitin sulfate A. In man, a deficiency of this enzymatic activity causes the lysosomal storage disorder, Maroteaux-Lamy disease (mucopolysaccharidosis Type VI; MPS VI). MPS VI in Siamese cats also has been described, and the comparative pathologic and biochemical abnormalities of the human and feline disorders have been well characterized. The present study describes the isolation and expression of cDNAs encoding feline ARSB and the assignment of the feline ARSB gene to feline chromosome A1. The full-length feline ARSB cDNA sequence is 1939 bp, including 3 and 328 bp of 5' and 3' untranslated sequences, respectively, and a 1608-bp open reading frame encoding 535 amino acids. The predicted human and feline ARSB proteins are 91% identical and 94% similar. However, despite this high homology, the predicted feline ARSB polypeptide has nine cysteine residues, while the human enzyme has eight. The presence of the extra cysteine residue at position 451 in the feline enzyme may explain why feline ARSB is a homodimer and the human enzyme is a monomer. To facilitate comparative structure/function studies of the human and feline enzymes and to initiate somatic gene therapy trials in the MPS VI cats, a full-length feline ARSB cDNA was reconstructed from a 1440-bp partial cDNA and an ARSB fragment amplified from feline first-strand cDNA by the polymerase chain reaction. The functional integrity of this cDNA was demonstrated by transient expression in human embryonic kidney cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Estimation of heterozygosity for single-probe multilocus DNA fingerprints.
In spite of the increasing application of DNA fingerprinting to natural populations and to the genetic identification of humans, explicit methods for estimation of basic population genetic parameters from DNA fingerprinting data have not been developed. Contributing to this omission is the inability to determine, for multilocus fingerprinting probes, relatively important genetic information, such as the number of loci, the number of alleles, and the distribution of these alleles into specific loci. One of the most useful genetic parameters that could be derived from such data would be the average heterozygosity, which has traditionally been employed to measure the level of genetic variation within populations and to compare genetic variation among different loci. We derive here explicit formulas for both the estimation of average heterozygosity at multiple hypervariable loci and a maximum value for this estimate. These estimates are based upon the DNA restriction-pattern matrices that are typical for fingerprinting studies of humans and natural populations. For several empirical data sets from our laboratory, estimates of average and maximal heterozygosity are shown to be relatively close to each other. Furthermore, variances of these statistics based on simulation studies are relatively small. These observations, as well as consideration of the effect of missing alleles and alternate numbers of loci, suggest that the average heterozygosity can be accurately estimated using phenotypic DNA fingerprint patterns, because this parameter is relatively insensitive to the lack of certain genetic information.
BEK, a receptor for multiple members of the fibroblast growth factor (FGF) family, maps to human chromosome 10q25.3----q26.
The gene for the fibroblast growth factor receptor BEK was assigned to human chromosome 10 by applying polymerase chain reaction techniques to DNAs from a panel of human x rodent somatic cell hybrids. The gene was further localized to 10q25.3----q26 by in situ hybridization.
Peroneal tendon subluxation in a case of anomalous peroneus brevis muscle.
We report a case of peroneal tendon subluxation as a result of an anomalous extension of the peroneus brevis muscle into the fibular groove, causing an encroachment phenomenon, stretching-out of the superior peroneal retinaculum, longitudinal splitting of the peroneus brevis tendon, subluxation of the peroneal tendons, and peroneal tenosynovitis. We describe a simple surgical technique for tendon stabilization, after decompression of the fibular groove.
Genomic dispersal of the ets gene family during metazoan evolution.
Evolutionary homologs of the ets proto-oncogene have been discovered in the genomes of widely divergent eucaryote species from Drosophila to sea urchin to vertebrates. The prototype mammalian ets-1 and ets-2 genes are divided into three coding domains that differ in their rate of accumulation of sequence divergence. An analysis of sequence divergence of ets gene homologs in various species has produced a phylogenetic history of the ets gene family in the context of metazoan evolutionary radiation. A minimum of five duplication events of ets primordial genes were evident, namely (1) a duplication that separates primitive ets genes (Drosophila precursor of 74E, mouse PU.1 and human ELK1) from the ets-1, ets-2, erg ancestor; (2) and (3) two duplications that established separate ets, erg and elg/GABP-alpha lineages which occurred prior to invertebrate-vertebrate divergence; (4) divergence of ets-1 and ets-2 gene family also associated with vertebrate-invertebrate divergence; (5) duplication of ets-1 and ets-2 in Xenopus laevis to produce two ets-1 genes and two ets-2 genes during genomic tetraploidation in the recent ancestry of this species.
Anatomy, histology, and vascularity of the glenoid labrum. An anatomical study.
We studied the gross, histological, and vascular anatomy of the glenoid labrum in twenty-three fresh-frozen shoulders from cadavera to demonstrate its cross-sectional anatomy, its microvascularity, and its attachments. The superior and anterosuperior portions of the labrum are loosely attached to the glenoid, and the macro-anatomy of those portions is similar to that of the meniscus of the knee. The superior portion of the labrum also consistently inserts directly into the biceps tendon, while its inferior portion is firmly attached to the glenoid rim and appears as a fibrous, immobile extension of the articular cartilage. The arteries supplying the periphery of the glenoid labrum come from the suprascapular, circumflex scapular, and posterior circumflex humeral arteries. In general, the superior and anterosuperior parts of the labrum have less vascularity than do the posterosuperior and inferior parts, and the vascularity is limited to the periphery of the labrum. Vessels supplying the labrum originate from either capsular or periosteal vessels and not from the underlying bone.
Red spells danger for whelk eaters.
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Linkage mapping of the human CSF2 and IL3 genes.
Interleukin 3 (encoded by the IL3 gene) and granulocyte-macrophage colony-stimulating factor (encoded by the CSF2 gene) are small secreted polypeptides that bind to specific cell surface receptors and regulate the growth, gene expression, and differentiation of many of the hematopoietic cell lineages, particularly nonlymphoid cells. The IL3 and CSF2 genes have been cloned and mapped to human chromosome bands 5q23-31. Only 10 kilobases of DNA separates the two genes, suggesting that they have a common origin and/or regulation. We have cloned 70 kilobases of genomic DNA that includes the IL3 and CSF2 genes, as well as flanking sequences, and report a physical map of this region. Several unique-sequence DNA segments have been identified in this region, and one of these fragments detects two restriction fragment length polymorphisms in DNA from unrelated Caucasians. Segregation of these DNA polymorphisms was followed in the Centre Etudé du Polymorphisme Humaine (CEPH) panel of 40 large three-generation pedigrees, and linkage was detected with 17 genetic markers previously typed in these families. Multipoint linkage analysis permits the placement of the region containing the IL3 and CSF2 structural genes on the recombination-genetic linkage map of chromosome 5q and thereby allows the role of these genes in leukemogenesis to be more critically examined.
Culture medium and protein supplementation influence in vitro fertilization and embryo development in the domestic cat.
The influence of culture medium and protein supplements on in vitro fertilization (IVF) and morula-to-blastocyst development in culture was examined in the domestic cat. In Study I, follicular oocytes were fertilized and cultured in 1) modified Krebs Ringer bicarbonate (mKRB); 2) modified Tyrode's solution (TALP) without phosphate or glucose; or 3) Ham's F10. All media contained bovine serum albumin (BSA). Fertilization rates were similar (P greater than .05) among mKRB (75.0%), TALP (70.6%), and Ham's F10 (80.0%) treatments. Compared to TALP (77.8%), more (P less than .05) embryos in Ham's F10 (95.0%) developed to the morula stage; development of mKRB embryos (88.9%) was intermediate and not different (P greater than .05). Study II evaluated the effects of protein availability and type on IVF and embryo growth. Ham's F10 was supplemented with polyvinylalcohol (PVA, 2 mg/ml), BSA (4 mg/ml), fetal calf serum (FCS, 5%), or estrous cat serum (ECS, 5%) and used to fertilize and culture embryos. Fertilization was enhanced (P less than .05) using either FCS (84.0%) or ECS (85.2%) compared to PVA (67.3%). Oocytes exposed to BSA fertilized at a rate (76.1%) similar (P greater than .05) to other treatments. Embryos exposed to all four treatment groups (PVA, 82.8%; BSA, 82.8%; FCS, 92.9%; or ECS, 97.8%) were equally capable (P greater than .05) of becoming morulae. However, more FCS- or ECS-supplemented morulae continued to the early blastocyst stage (30.8%, 22.2%, respectively, P less than .05) than PVA- (10.3%) or BSA- (13.8%) exposed morulae. In the domestic cat IVF system, the type of medium and protein supplement used appears to have a greater impact on embryo development in vitro than on fertilization.(ABSTRACT TRUNCATED AT 250 WORDS)
The Lake Casitas wild mouse: evolving genetic resistance to retroviral disease.
A small colony of feral mice from California continues to flourish in spite of a virulent epizootic of pathological retrovirus. Epidemiological and genetic studies revealed that the viral infection is strongly balanced by the polymorphic resistance locus, Fv-4, a transcriptionally active but truncated provirus that originated in the East Asian ancestors of the Californian mice. The natural history of these populations represents a graphic example of genomic adaptation in free-ranging populations to regulate and delimit infectious disease.
Linkage mapping of human polymorphic proteins identified by two-dimensional electrophoresis.
Nineteen polymorphic lymphocyte proteins were previously detected by two-dimensional protein electrophoresis (2DE). In this report, we describe the genetic linkage mapping of six of these polymorphic proteins (PNIA1-PNIA6), the identification by genetic linkage of a seventh (glyoxalase 1 on 6p21), and support for the mapping of an eighth (plastin or LCP1) to near the ESD locus on Chr 13. PNIA1-PNIA6 were assigned, respectively, to 10q26, 16p13.3, 10q, 11p15, 3q, and 19q13. These genetic linkages were achieved by classical linkage analysis of 2DE protein charge polymorphisms to the panel of RFLPs previously typed in nine pedigrees in the Centre D'Etude du Polymorphisme Humain (CEPH) collection.
Molecular cloning, chromosomal assignment, and nucleotide sequence of the feline homeobox HOX3A.
The feline homolog to the mammalian homeobox locus, HOX3A, was isolated by screening a domestic cat genomic library with the murine Hox-3.1 probe. The nucleotide sequence similarity of the feline homeobox was 96% to human HOX3A, 94% to mouse Hox-3.1, and 94% to rat R4. The deduced amino acid sequence (homeodomain) of this feline homeobox was identical to all homeodomains of these cognate genes. Using a panel of feline x rodent somatic cell hybrids, the HOX3A locus was assigned to feline chromosome B4. Human HOX3A and mouse Hox-3.1 have been mapped previously to human chromosome 12 and mouse chromosome 15, respectively, both of which share syntenic homology to feline chromosome B4. These data demonstrate evolutionary conservation of both HOX3A gene sequences and chromosomal location during mammalian evolution.
Mapping of the gene encoding the alpha subunit of the stimulatory G protein of adenylyl cyclase (GNAS1) to 20q13.2----q13.3 in human by in situ hybridization.
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Mammalian genome mapping: lessons and prospects.
The recent emphasis on human genome mapping has stimulated the development of gene maps in close to thirty mammalian species. Animal gene maps provide an invaluable resource for genetic analysis and manipulation of phenotypic characters, as well as a retrospective glimpse at the patterns and processes of genome evolution. An empirical strategy for developing new gene maps in mammals by emphasizing two important classes of index or anchor marker loci is presented.