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R L Stallings

Publications and source records attributed to R L Stallings.

At least 19 recordsLinked to original sources
PubMed1995

Primary structure of human lumican (keratan sulfate proteoglycan) and localization of the gene (LUM) to chromosome 12q21.3-q22.

A human corneal fibroblast cDNA library was screened with a bovine lumican cDNA probe to obtain three clones. Sequencing of the longest clone (1.75 kb) yielded an open reading frame of 1014 bp coding for a 338-amino-acid core protein. Amino acid sequencing of a tryptic peptide resulted in a 9-amino-acid match with the derived primary structure, confirming the identity of these clones. Human lumican displays all of the features of small interstitial proteoglycans: N- and C-terminal domains with highly conserved cysteines and a central domain containing nine repeats of slight variations of the leucine motif LXXLXLXXNXL. Like bovine lumican, the human core protein contains four possible N-glycosylation sites in the central domains, all or some of which are substituted with keratan sulfate side chains. At the amino acid level, it is 90% identical with bovine and 72% identical with the chicken core protein. The gene (LUM) was localized to human chromosome 12 by hybridizing a cDNA probe to a Southern blot containing a human/hamster monochromosomal mapping panel DNA. Further sublocalization to 12q21.3-q22 was performed by the fluorescence in situ hybridization technique using a lumican P1 genomic clone. By immunohistochemical staining, we show lumican's presence, not only in the corneal stroma as shown previously, but also in the dermal area of the skin, indicating a wider distribution of this proteoglycan.

Amino Acid Sequence↗
PubMed1995

Efficient pooling designs for library screening.

We describe efficient methods for screening clone libraries, based on pooling schemes that we call "random k-sets designs." In these designs, the pools in which any clone occurs are equally likely to be any possible selection of k from the v pools. The values of k and v can be chosen to optimize desirable properties. Random k-sets designs have substantial advantages over alternative pooling schemes: they are efficient, flexible, and easy to specify, require fewer pools, and have error-correcting and error-detecting capabilities. In addition, screening can often be achieved in only one pass, thus facilitating automation. For design comparison, we assume a binomial distribution for the number of "positive" clones, with parameters n, the number of clones, and c, the coverage. We propose the expected number of resolved positive clones--clones that are definitely positive based upon the pool assays--as a criterion for the efficiency of a pooling design. We determine the value of k that is optimal, with respect to this criterion, as a function of v, n, and c. We also describe superior k-sets designs called k-sets packing designs. As an illustration, we discuss a robotically implemented design for a 2.5-fold-coverage, human chromosome 16 YAC library of n = 1298 clones. We also estimate the probability that each clone is positive, given the pool-assay data and a model for experimental errors.

Binomial Distribution↗
PubMed1995

Conservation and evolution of (CT)n/(GA)n microsatellite sequences at orthologous positions in diverse mammalian genomes.

The distribution and evolution of (CT)n microsatellites were examined in GenBank mammalian DNA sequences because these microsatellites are known to play important roles in the regulation of some genes in Drosophila melanogaster. A total of 236 (CT)n microsatellite loci were found in GenBank mammalian gene sequences. To determine whether (CT)n microsatellite arrays were conserved at orthologous positions in distantly related mammalian sequences, we determined whether orthologous sequences existed in GenBank for each of the 236 loci. A total of 47 sequence alignments could be made. For rodent x rodent comparisons, 7 of 8 (CT)n arrays were conserved at identical positions in each pair of orthologous sequences. Comparisons of orthologous sequences between different orders of mammals indicated that 11 of 39 (CT)n arrays occurred at orthologous positions or within 1 kb of orthologous positions in each pair of sequences. It appears that there is some level of conservation of (CT)n repeats in distantly related mammals. However, this level of conservation may not be greater than what might be expected to occur by chance. In 13 cases where (CT)n arrays were not conserved at orthologous positions, the lack of a (CT)n array in one sequence resulted from either nucleotide substitution within an array or nonexpansion of a shorter (CT)n element. In these cases, significant sequence identity could be detected throughout the entire region even though the repeat array was not detected in one of the sequences. In contrast, there was a disruption of sequence identity in the (CT)n microsatellite region that ranged from 24 to 1600 bp in 21 cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A large duplicated area in the polycystic kidney disease 1 (PKD1) region of chromosome 16 is prone to rearrangement.

An area of 500 kb at the proximal end of the polycystic kidney disease 1 (PKD1) region has been mapped in detail, with 260 kb cloned in cosmids. The area cloned from normal individuals contains two homologous but divergent regions each of 75 kb, including the previously described marker 26-6. Pulsed-field gel electrophoresis identified a duplication of 75 kb of this region, referred to as the OX duplication (OXdup), in three patients with PKD1. The OXdup probably arose by an unequal exchange promoted by misalignment of partially homologous areas. Study of the OXdup in a large PKD1 family showed that it segregated with PKD1 in just one-half of the family, indicating that a recent crossover had occurred between the OXdup and PKD1 and showing that it was not a PKD1 mutation. Further analysis identified an OXdup breakpoint fragment: the OXdup was subsequently identified in 2 normal individuals of 110 assayed. The finding of the OXdup and in other individuals an 11-kb deletion (OXdel) at a similar point within this duplicated area indicates that this is an unusually unstable genomic region.

Chromosome Mapping↗
PubMed1994

FISH mapping of a human chromosome 16 constitutional pericentric inversion inv(16)(p13q22) found in a large kindred.

Fluorescence in situ hybridization analysis (FISH) was used to map the constitutional chromosome 16 pericentric inversion breakpoints inv(16)(p13q22) detected in one individual (II-2) from a large kindred [Bianchi et al., 1992: Am J Med Genet 43:791-795]. The breakpoints found in individual II-2 mapped to distinctly different locations than the chromosome 16 pericentric inversion breakpoints commonly acquired in acute nonlymphocytic leukemia. The constitutional pericentric inversion breakpoints also do not map to regions where low abundance repetitive DNA sequences found in bands 16p13 and q22 are located. The results indicate that low abundance, chromosome 16-specific repetitive DNA sequences in bands p13 and q22 are probably not causally related to the inversion that is found in many members of a large kindred [Bianchi et al., 1992].

Adult↗
PubMed1994

Distribution of trinucleotide microsatellites in different categories of mammalian genomic sequence: implications for human genetic diseases.

The distribution of all trinucleotide microsatellite sequences in the GenBank database was surveyed to provide insight into human genetic disease syndromes that result from expansion of microsatellites. The microsatellite motif (CAG)n is one of the most abundant microsatellite motifs in human GenBank DNA sequences and is the most abundant microsatellites found in exons. This fact may explain why (CAG)n repeats are thus far the predominant microsatellites expanded in human genetic diseases. Surprisingly, (CAG)n microsatellites are excluded from intronic regions in a strand-specific fashion, possibly because of similarity to the 3' consensus splice site, CAGG. A comparison of the positions of microsatellites in human vs rodent homologous sequences indicates that some arrays are not extensively conserved for long periods of time, even when they form parts of protein coding sequences. The general lack of conservation of trinucleotide repeat loci in diverse mammals indicates that animal models for some human microsatellite expansion syndromes may be difficult to find.

Animals↗
PubMed1994

In situ hybridization mapping of human chromosome 16: evidence for a high frequency of repetitive DNA sequences.

Fluorescence in situ hybridization (FISH) provides a rapid approach to regional localization of overlapping clone sets (contigs) developed by various fingerprinting approaches. We have used 70 cosmid clones derived from 48 different contigs, part of the developing contig map of chromosome 16 (Stallings et al., 1990, 1992a), to cytogenetically map an estimated 8.6 million base pairs (Mbp) of chromosome 16 DNA (approximately 8-9% total coverage). Although the majority of cosmid contigs hybridized to single sites on chromosome 16, a significant fraction (23%) hybridized to multiple regions on chromosome 16; a subset of these also hybridized to other human chromosomes. In most instances, clones that mapped to multiple locations were found to contain low-abundance repetitive DNA sequences. The FISH data presented here, coupled with published mapping data from somatic cell hybrids (Callen et al., 1992), permits independent verification of the integrity of chromosome 16 cosmid contigs. The order of clones derived by FISH agrees closely with the cell hybrid mapping data and can be correlated with chromosome bands and specific chromosomal translocation breakpoints.

Chromosome Mapping↗
PubMed1994

Evidence of linkage disequilibrium in the Spanish polycystic kidney disease I population.

Forty-one Spanish families with polycystic kidney disease 1 (PKD1) were studied for evidence of linkage disequilibrium between the disease locus and six closely linked markers. Four of these loci--three highly polymorphic microsatellites (SM6, CW3, and CW2) and an RFLP marker (BLu24)--are described for the first time in this report. Overall the results reveal many different haplotypes on the disease-carrying chromosome, suggesting a variety of independent PKD1 mutations. However, linkage disequilibrium was found between BLu24 and PKD1, and this was corroborated by haplotype analysis including the microsatellite polymorphisms. From this analysis a group of closely related haplotypes, consisting of four markers, was found on 40% of PKD1 chromosomes, although markers flanking this homogeneous region showed greater variability. This study has highlighted an interesting subpopulation of Spanish PKD1 chromosomes, many of which have a common origin, that may be useful for localizing the PKD1 locus more precisely.

Alleles↗
PubMed1993

Identification of yeast artificial chromosomes containing the inversion 16 p-arm breakpoint associated with acute myelomonocytic leukemia.

We report the cloning of the chromosome 16 p-arm breakpoint involved in inversion 16(p13;q22) associated with subtype of acute myelomonocytic leukemia (AMML) M4Eo. Inter-Alu polymerase chain reaction (PCR) products from a series of interspecific somatic cell hybrids that contain only small portions of the human chromosome 16 p-arm were generated for use as fluorescent in-situ hybridization (FISH) probes. When applied to patient cells, rapid and unambiguous identification of the inversion resulted. Using FISH analysis, cosmid clones associated with the hybrids were identified that bracketed the p-arm breakpoint. A repeat-free fragment of one of these cosmids (35B11) when used as probe on Southern blots from pulsed-field gels identified rearranged macrorestriction fragments in patient DNA. Yeast artificial chromosomes (YACs) were isolated using sequences derived from cosmids flanking 35B11 in a cosmid contig. Of 4 YACs so identified, 3 were shown by FISH to cross the inversion-16 p-arm breakpoint. Therefore, the breakpoint has been molecularly cloned, and identified as being within these 3 YACs. These clones will facilitate the unraveling of the genetic events associated with inversion-16 and are available tools with immediate clinical application.

Base Sequence↗
PubMed1993

Fine genetic mapping of the Batten disease locus (CLN3) by haplotype analysis and demonstration of allelic association with chromosome 16p microsatellite loci.

Batten disease, juvenile onset neuronal ceroid lipofuscinosis, is an autosomal recessive neurodegenerative disorder characterized by accumulation of autofluorescent lipopigment in neurons and other cell types. The disease locus (CLN3) has previously been assigned to chromosome 16p. The genetic localization of CLN3 has been refined by analyzing 70 families using a high-resolution map of 15 marker loci encompassing the CLN3 region on 16p. Crossovers in three maternal meioses allowed localization of CLN3 to the interval between D16S297 and D16S57. Within that interval alleles at three highly polymorphic dinucleotide repeat loci (D16S288, D16S298, D16S299) were found to be in strong linkage disequilibrium with CLN3. Analysis of haplotypes suggests that a majority of CLN3 chromosomes have arisen from a single founder mutation.

Alleles↗
PubMed1993

Identification and regional localization of a human IMP dehydrogenase-like locus (IMPDHL1) at 16p13.13.

Sequence-tagged sites (STSs) are versatile chromosomal markers for a variety of genome mapping efforts. In this report, we describe a randomly generated STS (323F4) from human chromosome 16 genomic DNA that has 90.0% sequence identity to the type I human inosine-5'-monophosphate dehydrogenase (IMPDH1) gene and 72% identity to the type II human inosine-5'-monophosphate dehydrogenase (IMPDH2) gene. Additional sequencing by primer walking has provided a total of 1380 bp of the human chromosome 16 sequence. The IMPDH-like sequence 323F4 was regionally localized by PCR analysis of a panel of somatic cell hybrids containing different portions of human chromosome 16 to 16p13.3-13.12, between the breakpoints found in hybrids CY196/CY197 and CY198. This regional mapping assignment was further refined to subband 16p13.13 by high-resolution fluorescence in situ hybridization using cosmid 323F4 as a probe. We conclude that a third, previously undescribed IMPDH locus, termed IMPDHL1, exists at human chromosome 16p13.13.

Animals↗
PubMed1992

Evaluation of a cosmid contig physical map of human chromosome 16.

A cosmid contig physical map of human chromosome 16 has been developed by repetitive sequence finger-printing of approximately 4000 cosmid clones obtained from a chromosome 16-specific cosmid library. The arrangement of clones in contigs is determined by (1) estimating cosmid length and determining the likelihoods for all possible pairwise clone overlaps, using the fingerprint data, and (2) using an optimization technique to fit contig maps to these estimates. Two important questions concerning this contig map are how much of chromosome 16 is covered and how accurate are the assembled contigs. Both questions can be addressed by hybridization of single-copy sequence probes to gridded arrays of the cosmids. All of the fingerprinted clones have been arrayed on nylon membranes so that any region of interest can be identified by hybridization. The hybridization experiments indicate that approximately 84% of the euchromatic arms of chromosome 16 are covered by contigs and singleton cosmids. Both grid hybridization (26 contigs) and pulsed-field gel electrophoresis experiments (11 contigs) confirmed the assembled contigs, indicating that false positive overlaps occur infrequently in the present map. Furthermore, regional localization of 93 contigs and singleton cosmids to a somatic cell hybrid mapping panel indicates that there is no bias in the coverage of the euchromatic arms.

Chromosome Banding↗
PubMed1992

High-resolution cytogenetic-based physical map of human chromosome 16.

A panel of 54 mouse/human somatic cell hybrids, each possessing various portions of chromosome 16, was constructed; 46 were constructed from naturally occurring rearrangements of this chromosome, which were ascertained in clinical cytogenetics laboratories, and a further 8 from rearrangements spontaneously arising during tissue culture. By mapping 235 DNA markers to this panel of hybrids, and in relation to four fragile sites and the centromere, a cytogenetic-based physical map of chromosome 16 with an average resolution of 1.6 Mb was generated. Included are 66 DNA markers that have been typed in the CEPH pedigrees, and these will allow the construction of a detailed correlation of the cytogenetic-based physical map and the genetic map of this chromosome. Cosmids from chromosome 16 that have been assembled into contigs by use of repetitive sequence fingerprinting have been mapped to the hybrid panel. Approximately 11% of the euchromatin is now both represented in such contigs and located on the cytogenetic-based physical map. This high-resolution cytogenetic-based physical map of chromosome 16 will provide the basis for the cloning of genetically mapped disease genes, genes disrupted in cytogenetic rearrangements that have produced abnormal phenotypes, and cancer breakpoints.

Animals↗
PubMed1992

CpG suppression in vertebrate genomes does not account for the rarity of (CpG)n microsatellite repeats.

Simple microsatellite repetitive sequences are widely distributed in eukaryotic genomes. Using the GCG Find program, the distribution of each type of mono- and dinucleotide repetitive sequence has been examined in GenBank sequences. Examples of each type of simple satellite sequence could be found, although the frequency of (CpG)n greater than or equal to 8 repeats was extremely low. The suppression of CpG dinucleotides in vertebrates does not adequately explain the rarity of this repeat since (CpG)n repeats are also extremely infrequent in species genomes where CpG dinucleotides are not suppressed. Instead, it is proposed that (CpG)n repeats must possess a DNA conformation that has a deleterious structural effect.

Animals↗
PubMed1992

Chromosome 16-specific repetitive DNA sequences that map to chromosomal regions known to undergo breakage/rearrangement in leukemia cells.

Human chromosome 16-specific low-abundance repetitive (CH16LAR) DNA sequences have been identified during the course of constructing a physical map of this chromosome. At least three CH16LAR sequences exist and they are interspersed, in small clusters, over four regions that constitute more than 5% of the chromosome. CH16LAR sequences were observed in one unusually large cosmid contig (number 55), where the ordering of clones was difficult because these sequences led to false overlaps between noncontiguous clones. Contig 55 contains 78 clones, or approximately 2% of all the clones contained within the present cosmid contig physical map. Fluorescent in situ hybridization of multiple clones, including cosmid and YAC contig 55 clones, mapped the four CH16LAR-rich regions to bands p13, p12, p11, and q22. These regions are of biological interest since the pericentric inversion and the interhomologue translocation breakpoints commonly found in acute nonlymphocytic leukemia (ANLL) subtype M4 fall within these bands. Sequence analysis of a 2.2-kb HindIII fragment from a cosmid containing a CH16LAR sequence indicated that one of the CH16LAR elements is similar to a minisatellite sequence in that the core repeat is only 40 bp in length. Additional characterization of other repetitive elements is in progress.

Animals↗
PubMed1991

A refined physical map of the long arm of human chromosome 16.

Mapping of 33 anonymous DNA probes and 12 genes to the long arm of chromosome 16 was achieved by the use of 14 mouse/human hybrid cell lines and the fragile site FRA16B. Two of the hybrid cell lines contained overlapping interstitial deletions in bands q21 and q22.1. The localization of the 12 genes has been refined. The breakpoints present in the hybrids, in conjunction with the fragile site, can potentially divide the long arm of chromosome 16 into 16 regions. However, this was reduced to 14 regions because in two instances there were no probes or genes that mapped between pairs of breakpoints.

Animals↗