Biomedical subjects
Publications and source records attributed to K M Reed.
Microsatellite loci continue to be important tools in genetic analysis of populations and experimental families. Twelve new microsatellite sequences for the turkey were obtained from a small insert M13 library. Polymorphism at these new loci was examined in both domestic and wild birds. Seven of 12 loci were polymorphic in domestic turkeys, whereas 10 loci were polymorphic in our sample of wild birds.
Chromosome-specific libraries aid in the development of genetic maps and focus marker development in areas of the genome with identified quantitative trait loci (QTL). A small-insert BTA29 library constructed by microdissection of a 1:29 Rb-fusion cell line, was screened for dinucleotide repeats (CA)(15) and/or (GA)(15) with the goal of generating new genetic markers for this, the smallest bovine autosome. A total of 90 primer pairs were designed and 82 of these successfully amplified bovine genomic DNA by PCR. In addition to these 82 loci, primer pairs were developed for nine putative genes identified from the sequenced clones by BLAST searches of GenBank. A somatic cell panel was used to test for synteny of the new loci with two previously mapped BTA29 markers located on the MARC bovine linkage map. A total of 75 of the 82 microsatellite (ms) loci were integrated into the MARC bovine linkage map. Linkage analysis placed 69 ms markers on BTA29, five on BTAX and one on BTA1. Combined results of the somatic cell and linkage analyses place 79 new markers (ms and gene-related) on BTA29, six loci on BTAX and two loci on BTA1. The results of this effort significantly increase the marker density on BTA29, expanding the ability to fine map QTL associated with this chromosome.
The locations and chromosomal characteristics of ribosomal DNA (rDNA) sites in the karyotypes of two extant North American species of mudminnows, Umbra pygmaea and U. limi (2n = 22, NF = 44), were analyzed sequentially by conventional Giemsa staining, Ag staining, CMA(3) fluorescence and fluorescence in situ hybridization (FISH). The nucleolar organizer regions (NORs) were located in the fourth chromosomal pair in both species (pericentromeric region in U. pygmaea and subtelomeric in U. LIMI). These sites were strongly CMA(3)-positive suggesting that the rDNA sites in these species are associated with GC-rich DNA. FISH with a rDNA probe gave consistently positive signals in the same regions detected by Ag-staining and CMA(3)-fluorescence. However, both species also had additional CMA(3)-positive/Ag-negative heterochromatic blocks at pericentrometric regions of several chromosomal pairs (three in U. pygmaea and five in U. limi). FISH revealed additional rDNA clusters in both species. It is hypothesized that a paracentric inversion of the chromosome arm carrying the NORs might be one of the rearrangements differentiating the karyotypes of two North American species. The presence of additional rDNA sites is indicative of more complex rearrangements. The pericentromeric NOR phenotype of Umbra pygmaea is similar to that seen in U. krameri and in the distantly related genus Esox.
The sex chromosome pair has been identified previously as the largest submetacentric pair in the genome in several species of the genus Salvelinus (eastern trouts and chars) including S. namaycush (lake trout) and as a large subtelocentric/acrocentric pair in several species of the genus Oncorhynchus (Pacific trouts and salmon). Sex chromosomes have not been identified in Salmo (Atlantic salmon and brown trout). Two paint probes, one specific for the short arm (Yp) and the other for the long arm (Yq) of the sex chromosome pair in Salvelinus namaycush were hybridized to chromosomes of Oncorhynchus mykiss (rainbow trout) and O. tshawytscha (chinook salmon) and Salmo salar (Atlantic salmon) and S. trutta (brown trout). The two probes hybridized to two different autosomal pairs in each of the Oncorhynchus species, supporting lack of homology between the sex chromosomes in the two genera. The Yp probe hybridized to interstitial regions on two different chromosome pairs in S. salar and one pair in S. trutta. The Yq probe hybridized to a different pair in both species.
Polymorphic genetic markers are important tools in the construction of comprehensive genetic maps. This paper reports on the isolation and characterization of 90 new bovine microsatellite (ms) loci from enriched genomic libraries. The sequence of one clone (locus MNB-85) showed significant similarity to an intron of the human promyelocytic leukemia zinc finger protein (PLZF) gene. Screening of bovine and porcine somatic cell panels places the bovine PLZF homolog on BTA-15 and the porcine PLZF homolog on SSC-9. The 90 new microsatellite loci increase the number of microsatellites available for cattle by >5%.
This paper reports on the development of a small-insert (approximately 700 bp) total-genomic library for sheep specifically designed for enrichment for microsatellite (ms) loci. Four enriched libraries were prepared by amplification of the primary library with CA15, CA11, TG15 and TG11 oligonucleotide primers. A total of 11,020 clones was recovered, screened for dinucleotide repeats and over 500 positive clones sequenced. Sequence analysis indicated low clone redundancy and yielded 105 new ovine ms loci. Seventy-two percent of the new loci were found to be polymorphic in the sires of the AgResearch International Mapping Flock (IMF). The 105 new microsatellite loci increase the number of microsatellites available for sheep by >7%.
This study examines sequence divergence in three spacer regions of the ribosomal DNA (rDNA) cistron, to test the hypothesis of unequal mutation rates. Portions of two transcribed spacers (ITS-1 and 5' ETS) and the non-transcribed spacer (NTS) or intergenic spacer (IGS) formed the basis of comparative analyses. Sequence divergence was measured both within an individual lake trout (Salvelinus namaycush) and among several related salmonid species (lake trout; brook trout, Salvelinus fontinalis; Arctic char, Salvelinus alpinus; Atlantic salmon, Salmo salar; and brown trout, Salmo trutta). Despite major differences in the length of the rDNA cistron within individual lake trout, minimal sequence difference was detected among cistrons. Interspecies comparisons found that molecular variation in the rDNA spacers did not conform to the predicted pattern of evolution (ITS spacers<ETS spacers<IGS). Specifically, the IGS contains a region that appears to be as highly, or more conserved than the ITS-1.
A total-genomic cosmid library was created to isolate complete copies of the rDNA cistron of lake trout (Salvelinus namaycush) in order to study the structure and organization of the intergenic spacer (IGS) in this species. A total of 60 rDNA-positive clones (average inserts > 25 kb) was recovered by screening the library with a rDNA-specific probe. Positive clones were assayed for the presence of the two internal rDNA spacers (ITS-1 and ITS-2) and the entire IGS fragment was successfully amplified from 42 clones by PCR. Length of the IGS fragments ranged from 9.4 to 17.8 kb. Comparative restriction mapping of the IGS-PCR products of several clones indicated two regions of extensive length variation surrounding a central region with sequence conservation. DNA sequence analysis was used to investigate the molecular basis of the IGS length variation and focused on identifying the region responsible for this variation. Over 9 kb of DNA sequence was obtained for one clone (A1) with a total IGS length of approximately 12.4 kb. Sequence of a conserved central region contained two open reading frames and a number of short direct repeats. Length variation in the IGS was determined by RFLP to result from differences in the number of copies of repetitive DNA sequences. These included an 89-bp tandem repeat (alpha repeats), an 82-bp element (beta repeats), a 168-177-bp element (chi repeats), and a 179-201-bp element (delta repeats). Overall nucleotide composition of the IGS was biased towards A and T (%GC = 47.4). Maintenance of discrete rDNA-length variants in lake trout suggests that the rate of gene conversion is insufficient to produce homogeneous copies across the genome.
The genome of the zebrafish, Danio rerio, contains two major classes of tandem repetitive elements (AT-rich and GC-rich). The AT-rich repeats can be further subdivided into two subgroups which differ by about 10% of 185 bp in the repeating unit. The chromosomal location of these sequences and the moderately repetitive 5S rDNA sequences was determined in two diploid zebrafish cell lines using in-situ hybridization with fluorochrome-labeled probes. The AT-rich sequences were found at the centromeres of all chromosome pairs and the GC-rich sequences were found in paracentromeric location on over half of the chromosomal pairs. Different patterns of hybridization were found for the two subgroups of the AT-rich family. One type hybridized primarily to centromeres of one half to two thirds of the chromosomal pairs and the other type to centromeres of about three fourths of the chromosomal pairs. The pattern of hybridization with the GC-rich sequences varied somewhat between the cell lines consistent with interindividual variation in the location of paracentromeric heterochromatin. The 5S rRNA genes are found on the long arm of chromosome 3. Most of this chromosome arm is late replicating, but apparently does not contain either the AT-rich or GC-rich repetitive sequences.
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Cross-species amplification of 520 chicken microsatellite markers was tested by polymerase chain reaction with genomic DNA of the turkey (Meleagris gallopavo). Each primer pair was tested at six different combinations of annealing temperature and MgCl2 concentration. A total of 280 (54%) of the primer pairs produced amplification products. The majority of these products were similar, if not identical in size to those expected based on the fragment sizes of the corresponding chicken loci. Structure of the dinucleotide repeat and flanking sequences was examined for 13 turkey fragments (amplified with chicken primers) and 5 chicken fragments (amplified with turkey primers). Sequence analysis found a wide array of mutations between species in addition to differences in repeat length. To estimate the usefulness of the amplified loci for genetic mapping in the turkey, allelic polymorphism was determined for 57 of the 280 amplified loci. A total of 20 of 57 markers (35%) were polymorphic with an average of 1.4 alleles per locus. The results of this study suggest that approximately 20% of the chicken microsatellite markers will be useful for mapping the turkey genome.
The chromosomes of longnose gar, Lepisosteus osseus, an extant representative of early radiation of actinopterygian fishes, were studied using conventional Giemsa-staining, Ag-staining, CMA3-fluorescence and fluorescence in-situ hybridization (FISH). The diploid chromosome number was 2n = 56 and the karyotype contained 11 pairs of metacentric, 6 pairs of submetacentric, 3 pairs of subtelocentric macrochromosomes and 16 microchromosomes. Nearly all macrochromosomes showed large CMA3-positive regions resembling the R-bands of higher vertebrates, indicating extensive distribution of GC-rich DNA along chromosomes. The nucleolar organizer regions (NORs) were located on the end of the short arm of a single small metacentric macrochromosomal pair. These sites were strongly CMA3-positive, suggesting that ribosomal sites are associated with GC-rich DNA. In-situ hybridization (FISH) with a rDNA probe gave consistently positive signals in the same regions detected by Ag-staining and CMA3-fluorescence. The evolutionary conservation of positive CMA3-fluorescence of ribosomal sites in 'holostean' and teleostean fishes is discussed.
As part of the University of Minnesota's initiative to map the turkey genome, we are currently evaluating chicken microsatellite loci for use in mapping the turkey genome. To date, 141 primer pairs have been tested for amplification at six different combinations of temperature and MgCl2 concentration. Microsatellite primer pairs from the Chicken Comprehensive Mapping Kit #2, and additional unpublished chromosome 1 and 2 primers were screened. Analyzable PCR products were produced from 78 of the 141 (55%) primer combinations. In the majority of cases (68%), PCR fragments obtained from the turkey were similar in size to respective chicken loci. The presence of dinucleotide repeats (CA/TG repeats) was determined by Southern hybridization with a (TG)15, oligonucleotide probe. Five of 12 (41.63%) turkey fragments hybridized under low stringency conditions. The length of the dinucleotide repeats in the turkey, relative to the chicken sequences, were found to correspond directly with hybridization intensity. Amplification of homologous loci was confirmed by direct sequencing and subsequent alignment of the turkey and chicken sequences. The results of this study indicate that the use of chicken-specific microsatellite primers will rapidly and significantly enhance construction of a genetic map for the turkey.
This study was designed to characterize further the nontranscribed intergenic spacers (NTSs) of the 5S rRNA genes of fish and evaluate this marker as a tool for comparative studies. Two members of the closely related North American Great Lakes cisco species complex (Coregonus artedi and C. zenithicus) were chosen for comparison. Fluorescence in situ hybridization found the ciscoes to have a single multicopy 5S locus located in a C band-positive region of the largest submetacentric chromosome. The entire NTS was amplified from the two species by polymerase chain reaction with oligonucleotide primers anchored in the conserved 5S coding region. Complete sequences were determined for 25 clones from four individuals representing two discrete NTS length variants. Sequence analysis found the length variants to result from presence of a 130-bp direct repeat. No two sequences from a single fish were identical. Examination of sequence from the coding region revealed two types of 5S genes in addition to pseudogenes. This suggests the presence of both somatic and germline (oocyte) forms of the 5S gene in the genome of Coregonus. The amount of variation present among NTS sequences indicates that accumulation of variation (mutation) is greater in this multicopy gene than is gene conversion (homogenization). The high level of sequence variation makes the 5S NTS an inappropriate DNA sequence for comparisons of closely related taxa.
Sequence variation in the control region (D-loop) of the mitochondrial DNA (mtDNA) was examined to assess the genetic distinctiveness of the shortjaw cisco (Coregonus zenithicus). Individuals from within the Great Lakes Basin as well as inland lakes outside the basin were sampled. DNA fragments containing the entire D-loop were amplified by PCR from specimens of C. zenithicus and the related species C. artedi, C. hoyi, C. kiyi, and C. clupeaformis. DNA sequence analysis revealed high similarity within and among species and shared polymorphism for length variants. Based on this analysis, the shortjaw cisco is not genetically distinct from other cisco species.
This work describes chromosomal localization, fine physical mapping, and population variation of the BglI element in the genome of Atlantic salmon (Salmo salar L.) and a similar sequence in the genome of brown trout (S. trutta L.). Results from a variety of complementary approaches, clearly demonstrate that the BglI element does not occur as a satellite-like repetitive DNA in these species but is part of the rDNA cistron as suggested by Goodier and Davidson (1993). Coincident hybridization of BglI clones with rDNA loci in both single and double-probe fluorescence in situ hybridization (FISH) experiments demonstrated physical linkage between the BglI element and rDNA loci. Fine physical mapping by Southern analysis and PCR amplification showed the BglI element to be located approximately 1.6 kb upstream of the 18S gene. The BglI element was used to screen for population-specific markers by Southern analysis. Population-specific banding patterns were only observed in brown trout, allowing identification of individual populations of this species. Sequence comparisons revealed sequences similar to the BglI element present in the rDNA cistron of other salmonids. This result suggests the presence of this sequence in the genome of the salmonid tetraploid ancestor.
The relationship between a 217-bp AluI fragment (SnAluI-33c) from lake trout (Salvelinus namaycush) which hybridizes to the nucleolar organizer regions (NORs) and the ribosomal RNA genes was examined by Southern analysis and comparative hybridization. Restriction enzymes with recognition sites mapped in the lake trout rDNA cistron were used to digest genomic DNA into fragments of predetermined size. Comparison of the hybridization pattern of SnAluI-33c with those of two rDNA-specific probes placed this fragment within the intergenic spacer region of the rDNA cistron, approximately 3 kb upstream (5') of the 18S gene. This finding is consistent with in situ hybridization experiments showing hybridization of this fragment to sites of rDNA [Reed, K.M. and Phillips, R.B., Cytogenet. Cell Genet. 70 (1995) 104-107]. Based on cross hybridization and sequence comparisons, homologous sequences are present in other salmonid species.
Polymorphism of the nucleolus organizer region (NOR) on the putative sex chromosomes of Arctic char (Salvelinus alpinus) was examined using conventional cytogenetic and molecular techniques. Variation was observed in the number, size and position of rDNA loci on the sex pair. Fluorescence in situ hybridization (FISH) analyses showed that the sex chromosomes of Arctic char lack the repetitive DNA sequences (Mbol/BglII family) that are a prominent feature of the sex chromosomes of lake trout (S. namaycush). Southern analyses of genomic DNAs using an rDNA fragment as probe revealed extensive restriction fragment length polymorphism (RFLP) variation among individuals. Despite the presence of variation in all aspects of this rDNA locus, no sex-specific differences were detected. Repetitive DNAs (multicopy rDNA as in Arctic char or tandem repetitive DNA as in lake trout) appear to play important but different roles in the evolution of the sex chromosomes in these species.