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J L Slightom

Publications and source records attributed to J L Slightom.

At least 19 recordsLinked to original sources
PubMed1997

Evolution of a fetal expression pattern via cis changes near the gamma globin gene.

One basis for the evolution of organisms is the acquisition of new temporal and spatial domains of gene expression. Such novel expression domains could be generated either by cis sequence changes that alter the complement of trans-acting regulators binding to control elements or by changes in the expression patterns of one or more of the regulatory (trans) factors themselves. The gamma globin gene is a prime example of a gene that has undergone a distinct change in temporal expression at a defined time in evolution. Approximately 35-55 million years ago, the previously embryonic gamma gene acquired a fetal expression pattern. This change occurred in a simian primate ancestor after the separation of simian and prosimian primates but before the further separation of the major simian lineages; thus, the (prosimian) galago gamma gene retains the ancestral embryonic expression pattern, whereas the (simian) human gamma gene is fetal. This analysis of galago and human gamma genes in transgenic mice demonstrates that cis changes in sequences within a 4.0-kilobase region surrounding the gamma gene were responsible for the evolution of a novel fetal expression pattern in the gamma globin genes of simian primates.

Animals↗
PubMed1997

Phylogenetic footprinting of hypersensitive site 3 of the beta-globin locus control region.

Hypersensitive site 3 (HS3) of the beta-like globin locus control region has been implicated as an important regulator of the beta-like globin genes, but the trans factors that bind HS3 have only been partially characterized. Using a five-species alignment (human, galago, rabbit, goat, and mouse) that represents 370 million years of evolution, we have identified 24 phylogenetic footprints in the HS3 core and surrounding regions. Probes corresponding to the human sequence at each footprint have been used in binding studies to identify the nuclear factors that bind within and near these conserved sequence elements. Among the high-affinity interactions observed were several binding sites for proteins with repressor activity, including YY1, CCAAT displacement protein, and G1/G2 complexes (uncharacterized putative repressors) and several binding sites for the stage selector protein. To complement this analysis, orthologous galago sequences were also used to derive probes and the pattern of proteins binding to human and galago probes was compared. Binding interactions differing between these two species could be responsible for the different expression patterns shown by the two gamma genes (galago gamma is embryonic; human gamma is fetal). Alternatively, binding interactions that are conserved in the two species may be important in the regulation of common expression patterns (eg, repression of gamma in adult life).

Animals↗
PubMed1997

Nucleotide sequence analysis of the human KCNJ1 potassium channel locus.

Detailed analyses of transcripts encoding various isoforms of the human potassium (K+, inward rectifying) channel ROM-K (also referred to as K(ir)1.1) revealed the existence of at least five distinct transcripts [Shuck et al., J. Biol. Chem. 269 (1994) 24261-24270]. These five hROM-K transcripts appear to be the result of alternative splicing of five exons. The nucleotide sequence of the genomic DNA including and spanning these exons (the KCNJ1 locus) was obtained directly from lambda and P1 clones (a total of 40 kb). The organization of the hKCNJ1 gene was determined by combining this sequence information with data obtained from primer extension and RT-PCR experiments. It appears that the hKCNJ1 gene utilizes multiple promoters, with promoter-like elements found 5' of exons 1, 4, or 5. The promoter 5' of exon 5 was unexpected; thus, it appears that the hKCNJ1 gene is capable of producing six distinct hROM-K transcripts via the use of three promoters and alternative splicing of five exons. Comparisons of the rat and human ROM-K cDNA sequences find human homologs (orthologs) for two of the three distinct rROM-K transcripts. A search of the complete human KCNJ1 sequence with the exon sequence that defines the other rROM-K transcript located a region of shared nucleotides, a putative sixth exon, in the hKCNJ1 gene. This finding suggests that the rKCNJ1 gene may contain an exon that is no longer or infrequently used in transcripts derived from the hKCNJ1 gene.

Amino Acid Sequence↗
PubMed1997

Intracellular IL-1 receptor antagonist promoter: cell type-specific and inducible regulatory regions.

The objective of these studies was to examine the molecular mechanisms involved in transcriptional regulation of the gene for the intracellular structural variant of the IL-1 receptor antagonist (icIL-1Ra) molecule. By reverse transcription-PCR analysis, constitutive expression of endogenous icIL-1Ra mRNA was observed in the epithelial cell lines A431 and HT-29, but not in the macrophage cell lines RAW 264.7 and U937, or in the lymphocyte cell lines Raji and Jurkat. However, icIL-1Ra mRNA expression was observed in response to stimulation with LPS in RAW 264.7 cells and to PMA and LPS in U937 cells. To examine the mechanisms of transcriptional regulation, 4.5 kb of the 5' flanking sequence was isolated from the human icIL-1Ra gene, sequenced, cloned into a luciferase expression vector (pIC4525.Luc), and examined in transfection studies. The pIC4525.Luc construct exhibited a pattern of expression in epithelial and macrophage cell lines similar to that of the endogenous icIL-1Ra gene. To obtain a generalized map of cell type-specific and inducible cis-acting DNA elements, nested 5' deletional mutants of the icIL-1Ra promoter were constructed. Results from transfection studies with these icIL-1Ra promoter/luciferase fusion constructs indicated that constitutive expression in epithelial cells was under the control of three positively acting regions located between bases -4525 to -1438, -288 to -156, and -156 to -49. In contrast, basal expression of pIC4525.Luc in transfected but unstimulated RAW 264.7 cells was under the control of a weak inhibitory region located between bases -4525 to -1438 and a strong positive element between -156 and -49. LPS induction of icIL-1Ra transcription in RAW 264.7 cells was regulated by strong positively acting DNA regions between bases -1438 to -909 and -156 to -49. In summary, the proximal region of the icIL-1Ra promoter, between bases -156 to -49, contains positive cis-acting elements that are needed for expression in both epithelial and monocyte cell lines. However, our results indicate that the ability of this proximal promoter region to control expression is strongly influenced, both positively and negatively, by other upstream cis-acting elements in a cell type-specific manner.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of two K+ inward rectifier (Kir) 1.1 potassium channel homologs from human kidney (Kir1.2 and Kir1.3).

The DNA sequence encoding the rat brain inward rectifier-10 K+ channel was amplified from rat brain RNA using reverse transcription-polymerase chain reaction and used to clone the human homolog. Low stringency screening of a human kidney cDNA library and subsequent DNA sequence analysis identified two related K+ inward rectifier cDNAs, referred to as Kir1.2 and Kir1.3, which were derived from transcription of distinct human genes. Kir1.2 represents the human homolog of the rat BIRK-10 sequence, whereas Kir1.3 was unique compared with all available sequence data bases. The genes that encode Kir1.2 and Kir1.3 were mapped to human chromosomes 1 and 21, respectively. Both genes showed tissue-specific expression when analyzed by Northern blots. Kir1.2 was only detected in brain >> kidney and was detected at high levels in all brain regions examined. Kir1.3 was most readily detected in kidney and was also expressed in pancreas > lung. Comparative analysis of the predicted amino acid sequences for Kir1.2 and Kir1.3 revealed they were 62% identical. The most remarkable difference between the two polypeptides is that the Walker Type A consensus binding motif present in both Kir1.1 and Kir1.2 was not conserved in the Kir1.3 sequence. Expression of the Kir1.2 polypeptide in Xenopus oocytes resulted in the synthesis of a K+-selective channel that exhibited an inwardly rectifying current-voltage relationship and was inhibited by external Ba2+ and Cs+. Kir1.2 current amplitude was reduced by >85% when the pH was decreased from pH 7.4 to 5.9 using the membrane-permeant buffer acetate but was relatively unaffected when pH was similarly lowered using membrane-impermeant biphthalate. The inhibition by intracellular protons was voltage-independent with an IC50 of pH 6.2 and a Hill coefficient of 1.9, suggesting the cooperative binding of 2 protons to the intracellular face of the channel. In contrast, Kir1.3 expression in Xenopus oocytes was not detectable despite the fact that the cRNA efficiently directed the synthesis of a polypeptide of the expected Mr in an in vitro translation system. Co-expression of Kir1.3 with either Kir1.1 or Kir1.2 reduced currents resulting from expression of these inward-rectifier subunits alone, consistent with a dominant negative influence on Kir1.1 and Kir1.2 expression.

Amino Acid Sequence↗
PubMed1997

The complete sequences of the galago and rabbit beta-globin locus control regions: extended sequence and functional conservation outside the cores of DNase hypersensitive sites.

The locus control region (LCR) of mammalian beta-globin genes covers at least 17 kb at the 5' end of the gene cluster and has been implicated in chromatin domain opening, enhancement, and insulation from neighboring sequences. Functional dissection of the LCR has defined the minimal cores for four of the five major DNase hypersensitive sites (HSs) that mark this regulatory region. To examine fully the patterns of conserved sequences in the mammalian homologs to the beta-globin LCR, we determined the complete DNA sequence of the galago beta-globin LCR and completed previously unsequenced regions of the rabbit LCR. Simultaneous alignment of these sequences with the human, goat, and mouse LCRs revealed conserved sequences (phylogenetic footprints) detected using three largely independent methods. The most highly conserved segments are found both within the HS cores and in some but not all regions flanking the cores. These results argue for an extended pattern of well-conserved sequences, many of which lie outside the minimal cores, and we show that a key sequence required for domain opening by the region including HS3 maps about 1 kb 5' to the minimal core. Differential phylogenetic footprints, containing sequences conserved in nonhuman mammals but not in humans, are found primarily around HS3, consistent with some species-specific differences in function that may be important for differences in hemoglobin switching during development.

Animals↗
PubMed1996

Reduction of two functional gamma-globin genes to one: an evolutionary trend in New World monkeys (infraorder Platyrrhini).

Nucleotide sequences were determined for the gamma1- and gamma2-globin loci from representatives of the seven anciently separated clades in the three extant platyrrhine families (Atelidae, Pitheciidae, and Cebidae). These sequences revealed an evolutionary trend in New World monkeys either to inactivate the gamma1 gene or to fuse it with the gamma2 gene, i.e. to have only one functional fetally expressed gamma gene. This trend is clearly evident in six of the seven clades: (i) it occurred in atelids by deletion of most of the gamma1 gene in the basal ancestor of this clade; (ii-iv) in pitheciid titi, saki, and cebid capuchin monkeys by potentially debilitating nucleotide substitutions in the proximal CCAAT box of the gamma1 promoters and (v and vi) in cebid owl and squirrel monkeys by crossovers that fused 5' sequence from gamma1 with 3' sequence from gamma2. In the five clades with gamma1 and gamma2 loci separated by intergenic sequences (the fifth clade being the cebid marmosets), the gamma2 genes retained an unaltered proximal CCAAT motif and their gamma2 promoters accumulated fewer nucleotide substitutions than did the gamma1 promoters. Thus, phylogenetic considerations indicate that the stem platyrrhines, ancestral to all New World monkeys, had gamma2 as the primary fetally expressed gamma gene. A further inference is that when the earlier stem anthropoid gamma gene duplicated, gamma2 (at its greater downstream distance from epsilon) could evade embryonic activation by the locus control region but could be fetally activated once released by regulatory mutations from fetal repressors.

Animals↗
PubMed1996

Evolutionary strategies for the elucidation of cis and trans factors that regulate the developmental switching programs of the beta-like globin genes.

We describe three strategies for the identification of specific cis and trans factors that regulate globin gene expression, all three of which are based on the evolution of the globin genes and their expression patterns. The first approach, phylogenetic footprinting, relies on a search for sequence similarities and is designed to elucidate the factors that control those expression patterns which are shared by orthologous globin genes of all eutherian mammals (e.g., the expression of the epsilon globin genes in the embryonic yolk sac and its repression in fetal and adult hematopoietic tissues). The second approach, differential phylogenetic footprinting, relies on a search for sequence differences. This approach may be of value in identifying the mechanisms underlying the generation of novel expression patterns in specific lineages (e.g., the expression of gamma as a fetal gene in the simian primates in contrast with the embryonic expression of gamma in all other mammals). Finally, motif-based phylogenetic analysis takes into consideration the fact that many transcription factors are quite flexible in the recognition of their cognate sites. The approach allows the detection of functionally conserved binding sites despite their sequence variation.

Adult↗
PubMed1996

Organization of a human UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase gene and a related processed pseudogene.

We have previously characterized a cDNA that encodes a full length human UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase) (J.A. Meurer et al., J. Biochem., 118, 568-574, 1995). The present report describes the characterization of the corresponding human GalNAc-transferase gene and a related pseudogene. Two human genomic libraries, lambda and P1, were screened with probes derived from the human GalNAc-transferase nucleotide sequence, resulting in the isolation of four genomic clones. Southern blotting, PCR analysis, and sequencing revealed that three clones, lambda.HG-5, P1.GALN-A, and P1.GALN-B, contained overlapping genomic sequences that encompass over 55 kilobase pairs (kb) of genomic DNA and comprise a portion of the human GalNAc-transferase 5'-and 3'-untranslated regions and the entire coding region. The human GalNAc-transferase gene structure consists of at least 11 exons ranging in size from 99 to > 620 nucleotides which are separated by 10 introns ranging in size from 0.7 to approximately 12.5 kb. The fourth genomic clone, P1-GALN-psi, contained a approximately 2.4 kb sequence region which shares an overall 78.6% nucleotide identity with coding region exons 1 and 3 through 11 of the human GalNAc-transferase gene. However, a lack of intron sequences, as well as the presence of multiple nucleotide mutations, insertions, and deletions that disrupt the potential GalNAc-transferase reading frame, suggest that P1.GALN-psi contains a processed pseudogene. Screening of a human/rodent somatic cell hybrid panel with a P1.GALN-psi probe localized the GalNAc-transferase pseudogene to chromosome 3. Hence, the human genome contains at least two related GalNAc-transferase genes that are located on separate chromosomes.

Base Sequence↗
PubMed1996

Differential expression of dystrophin isoforms in strains of mdx mice with different mutations.

Mutations in the dystrophin gene are responsible for Duchenne and Becker muscular dystrophy (DMD/BMD). Studies of dystrophin expression and function have benefited from use of the mdx mouse, an animal model for DMD/BMD. Here we characterized mutations in three additional strains of mdx mice, the mdx2cv, mdx4cv and mdx5cv alleles. The mutation in the mdx2cv mouse was found to be a single base change in the splice acceptor sequence of dystrophin intron 42. This mutation leads to a complex pattern of aberrant splicing that generates multiple transcripts, none of which preserve the normal open reading frame. In the mdx5cv allele, the dystrophin mRNA contains a 53 bp deletion of sequences from exon 10. Analysis of the genomic DNA uncovered a single A to T transversion in exon 10. Although this base change does not alter the encoded amino acid, a new splice donor was created (GTGAG) that generates a frameshifting deletion in the processed mRNA. In the mdx4cv allele, direct sequencing revealed a C to T transition in exon 53, creating an ochre codon (CAA to TAA). The differential location of these mutations relative to the seven known dystrophin promoters results in a series of mdx mouse mutants that differ in their repertoire of isoform expression, such that these mice should be useful for studies of dystrophin expression and function. The mdx4cv and mdx5cv strains may be of additional use in gene transfer studies due to their low frequency of mutation reversion.

Alleles↗
PubMed1995

High affinity YY1 binding motifs: identification of two core types (ACAT and CCAT) and distribution of potential binding sites within the human beta globin cluster.

PCR-assisted binding site selection was used to define the sequence characteristics of high affinity YY1 binding sites. Compilation of the sequences of 189 selected oligonucleotides containing high affinity YY1 binding sites revealed two types of core sequence: ACAT and CCAT. ACAT cores were surrounded by other invariant nucleotides, forming the consensus GACATNTT. A search of the 73 kb human beta-like globin cluster with this consensus revealed eight matching motifs, six of which were located within 1-3 kb upstream of the gamma and beta genes. CCAT-type cores were more variable in surrounding sequence context; the consensus VDCCATNWY was found to fit 89% of the selected CCAT-containing oligonucleotides. A search of the human beta globin cluster with CCAT consensus sequences revealed 171 potential YY1 binding sites. Several of these were tested directly in gel shift assays and confirmed as high affinity YY1 binding sites. Finally, a strategy called motif-based phylogenetic analysis was employed to determine which of the 179 total sites are evolutionarily conserved. This analysis permits the detection of functionally conserved binding sites despite sequence differences present between the two species. The 21 conserved sites identified will serve as important starting points in further dissection of the possible role of YY1 in globin gene regulation.

Animals↗
PubMed1995

Cloning and comparative sequence analysis of the gene encoding canine intercellular adhesion molecule-1 (ICAM-1).

Canine intercellular adhesion molecule-1 (ICAM-1) plays a primary role in the adherence of canine neutrophils to endothelial cells and in the cytotoxicity of canine neutrophils for adult cardiac myocytes. We have cloned the canine ICAM-1 gene and have analyzed the conservation of ICAM-1 amino acid (aa) sequences in man, chimpanzee, mouse, rat and dog. Canine ICAM-1 displays 61% identity with human ICAM-1. Cys residues critical to the immunoglobulin (Ig) fold structure and four sites of N-linked glycosylation are absolutely conserved in ICAM-1 from all species. Residues in the cytoplasmic tail associated with cytoskeletal alpha-actinin binding are highly conserved, supporting the hypothesis that intracellular attachment is indeed important for ICAM-1 function. Residues critical for human ICAM-1 binding to the beta 2-integrin leukocyte-function-associated antigen 1 (LFA-1) are highly conserved between all species, whereas those residues demonstrated to play an important role in interaction of human ICAM-1 with macrophage activation complex 1 (Mac-1) are not highly conserved. Residues critical for ICAM-1 binding to rhinovirus and malaria-infected red blood cells (IRBC) are not highly conserved.

Amino Acid Sequence↗
PubMed1995

Cloning of a canine cDNA homologous to the human transforming growth factor-beta 1-encoding gene.

A 1369-bp cDNA that encodes a homolog of the human transforming growth factor-beta 1 (TGF-beta 1) has been isolated from canine endothelial cells using a combination of PCR and traditional plaque-screening methods. The deduced 390-amino-acid sequence of the canine TGF-beta 1 precursor has 91-94% identity to those deduced from the previously described human and mouse TGF-beta 1 clones.

Amino Acid Sequence↗
PubMed1995

Fate of a redundant gamma-globin gene in the atelid clade of New World monkeys: implications concerning fetal globin gene expression.

Conclusive evidence was provided that gamma 1, the upstream of the two linked simian gamma-globin loci (5'-gamma 1-gamma 2-3'), is a pseudogene in a major group of New World monkeys. Sequence analysis of PCR-amplified genomic fragments of predicted sizes revealed that all extant genera of the platyrrhine family Atelidae [Lagothrix (woolly monkeys), Brachyteles (woolly spider monkeys), Ateles (spider monkeys), and Alouatta (howler monkeys)] share a large deletion that removed most of exon 2, all of intron 2 and exon 3, and much of the 3' flanking sequence of gamma 1. The fact that two functional gamma-globin genes were not present in early ancestors of the Atelidae (and that gamma 1 was the dispensible gene) suggests that for much or even all of their evolution, platyrrhines have had gamma 2 as the primary fetally expressed gamma-globin gene, in contrast to catarrhines (e.g., humans and chimpanzees) that have gamma 1 as the primary fetally expressed gamma-globin gene. Results from promoter sequences further suggest that all three platyrrhine families (Atelidae, Cebidae, and Pitheciidae) have gamma 2 rather than gamma 1 as their primary fetally expressed gamma-globin gene. The implications of this suggestion were explored in terms of how gene redundancy, regulatory mutations, and distance of each gamma-globin gene from the locus control region were possibly involved in the acquisition and maintenance of fetal, rather than embryonic, expression.

Alouatta↗
PubMed1995

ROMK inwardly rectifying ATP-sensitive K+ channel. II. Cloning and distribution of alternative forms.

The rat ROMK gene encodes inwardly rectifying, ATP-regulated K+ channels [K. Ho, C. G. Nichols, W. J. Lederer, J. Lytton, P. M. Vassilev, M. V. Kanazirska, and S. C. Hebert. Nature Lond. 362: 31-38, 1993; H. Zhou, S. S. Tate, and L. G. Palmer. Am. J. Physiol. 266 (Cell Physiol. 35): C809-C824, 1994], and mRNA encoding these channels is widely expressed in distal cortical and outer medullary nephron segments [see companion study; W.-S. Lee and S. C. Hebert. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F1124-F1131, 1995]. Using approaches based on homology to ROMK1, we have identified two additional ROMK isoforms, ROMK2b and ROMK3. Analysis of the nucleotide sequences of the ROMK isoforms indicates that molecular diversity of ROMK transcripts is due to alternative splicing at both the 5'-coding and 3'-noncoding regions. The splicing at the 5' end of ROMK gives rise to channel proteins with variable-length NH2 termini containing different initial amino acid sequences. Functional expression of these isoforms in Xenopus oocytes showed that they form functional Ba(2+)-sensitive K+ channels. The nephron distribution of mRNAs encoding alternatively spliced isoforms of ROMK (ROMK1-ROMK3) was investigated by reverse transcription-polymerase chain reaction (RT-PCR) of nephron segments dissected from rat kidney. Nondegenerate PCR primer pairs were designed to span at least one intron and to amplify specific alternatively spliced forms of ROMK.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Fluorescence-based cycle sequencing with primers selected from a nonamer library.

We have previously demonstrated that nonamers can prime manual T7 polymerase sequencing reactions. We next wanted to determine whether nonamers could be used to prime sequencing reactions for the Applied Biosystems Model 373A fluorescence-based sequencer. Both the Applied Biosystems T7 and Taq DNA Polymerase DyeDeoxy Terminator methodologies were tested, and successful results were obtained using a modified Taq DNA polymerase cycle sequencing procedure. The best nonamer cycle sequencing reaction conditions found, thus far, include denaturation at 96 degrees C for 30 s, primer-template annealing at 20 degrees C for 5 min, a 5-min ramp to the extension temperature, extension at 60 degrees C for 4 min and repeating this cycle 50 times. Furthermore, we found that the results were greatly improved by using linear (restriction enzyme cut) and alkaline-denatured, double-stranded DNA in combination with a doubling (2x) of the standard cycle sequencing reaction mixture containing a 2% final concentration of dimethyl sulfoxide. A total of 121 nonamer primers were tested, and approximately 50% primed successful cycle sequencing reactions. An average reading length of 257 bp was obtained from the successful reactions.

DNA Primers↗
PubMed1994

Cloning and characterization of multiple forms of the human kidney ROM-K potassium channel.

The rat kidney ROM-K1 potassium channel cDNA was used to clone the homolog from human kidney using a combination of cDNA cloning, reverse transcriptase-polymerase chain reaction (RT-PCR), and primer extension cloning methods. In addition to the human species homolog of ROM-K1, four additional transcripts that are formed by alternative splicing of a single human gene were also characterized (hROM-K2 to hROM-K5). All five transcripts share a common 3' exon that encodes the majority of the channel protein and in three of the isoforms translation is initiated at a start codon contained within this exon (hROM-K2, hROM-K4, and hROM-K5). The two other transcripts contain additional exons that potentially extend the open reading frame by either 19 amino acid residues (hROM-K1) or by 17 amino acid residues (hROM-K3). Comparison of the translation products from the three representative transcripts (hROM-K1, hROM-K2, and hROM-K3) confirmed that hROM-K1 gave the largest product (41.6 kDa) and was translated more efficiently than either hROM-K2 or hROM-K3. Also, despite the presence of several additional canonical acceptor sites for Asn-linked glycosylation relative to rat ROM-K1, all three channel polypeptides were glycosylated to a similar extent in the in vitro translation reactions when canine pancreatic microsomes were included. A survey of the tissue distribution of expression of the various forms in selected human tissues showed that the core-exon linked to all four possible 5' exons are detected almost exclusively in kidney. The core-exon was also detected in human kidney and lower amounts were detected in skeletal muscle > pancreas > spleen > brain = heart > liver RNAs by RT-PCR. Alternatively, Northern blot analysis of poly(A)+ RNAs from these same tissues revealed a 2.8-kilobase transcript only in kidney. Heterologous expression of either the hROM-K1, hROM-K2, or hROM-K3 channel transcripts in Xenopus oocytes led to the expression of K(+)-selective, Ba(2+)-sensitive inwardly rectifying channels as measured by whole cell currents. At this level of analysis, the channel properties of the individual forms could not be distinguished.

Amino Acid Sequence↗