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J D Freeman

Publications and source records attributed to J D Freeman.

At least 19 recordsLinked to original sources
PubMed2003

A SAGE approach to discovery of genes involved in autophagic cell death.

Programmed cell death (PCD), important in normal animal physiology and disease, can be divided into at least two morphological subtypes, including type I, or apoptosis, and type II, or autophagic cell death. While many molecules involved in apoptosis have been discovered and studied intensively during the past decade, autophagic cell death is not well characterized molecularly. Here we report the first comprehensive identification of molecules associated with autophagic cell death during normal metazoan development in vivo. During Drosophila metamorphosis, the larval salivary glands undergo autophagic cell death regulated by a hormonally induced transcriptional cascade. To identify and analyze the genes expressed, we examined wild-type patterns of gene expression in three predeath stages of Drosophila salivary glands using serial analysis of gene expression (SAGE) [7]. 1244 transcripts, including genes involved in autophagy, defense response, cytoskeleton remodeling, noncaspase proteolysis, and apoptosis, were expressed differentially prior to salivary gland death. Mutant expression analysis indicated that several of these genes were regulated by E93, a gene required for salivary gland cell death. Our analyses strongly support both the emerging notion that there is overlap with respect to the molecules involved in autophagic cell death and apoptosis, and that there are important differences.

Animals↗
PubMed2001

Evolution of natural killer cell receptors: coexistence of functional Ly49 and KIR genes in baboons.

Natural killer (NK) cells represent an important first line of defense against viruses and malignancy [1]. NK cells express a variety of inhibitory and activating receptors that interact with classical major histocompatibility complex (MHC) class I molecules on potential target cells and determine the NK cell response [2-4]. Mouse NK receptors are encoded by the C-type lectin multigene family Ly49. However, in humans, a completely different family of receptors, the immunoglobulin-like killer inhibitory receptors (KIRs), performs the same function [2-4]. One Ly49-like gene, Ly49L, exists in humans but is incorrectly spliced and assumed to be nonfunctional [5, 6]. Mouse KIR-like genes have not been found, and evidence suggests that the primate KIRs amplified after rodents and primates diverged [7, 8]. Thus, two structurally dissimilar families, Ly49 and KIR, have evolved to play similar roles in mouse and human NK cells. This apparent example of functional convergent evolution raises several questions. It is unknown, for example, when the Ly49L gene became nonfunctional and if this event affected the functional evolution of the KIRs. The distribution of these gene families in different mammals is unstudied, and it is not known if any species uses both types of receptors. Here, we demonstrate that the Ly49L gene shows evidence of conservation in other mammals and that the human gene likely became nonfunctional 6-10 million years ago. Furthermore, we show that baboon lymphocytes express both full-length Ly49L transcripts and multiple KIR genes.

Alternative Splicing↗
PubMed2001

Comparative analysis of the promoter regions and transcriptional start sites of mouse Ly49 genes.

Despite numerous studies on the function of Ly49 natural killer cell receptors in the mouse, relatively little is known about how these genes are regulated at the transcriptional level. In the present study, we sequenced and compared 800 bp of the promoter region of nine Ly49 genes from C57B1/6 mice. This comparison showed that there is a high degree of sequence identity between the genes, and also revealed a region which is conserved between the mouse genes and the human Ly49L gene, indicating a potential core promoter region. This analysis also found that Ly49B and H differ from the other genes in having long interspersed repetitive sequence in their promoter region which suggests a gene conversion or rearrangement involving these two genes. In addition, we performed 5' rapid amplification of cDNA ends on four Ly49 genes to localize transcriptional start sites. These experiments showed that the transcriptional initiation sites are heterogeneous for all of the genes examined, and that a large majority of Ly49G transcripts originate from the second exon as well as its first intron. Although potential TATA boxes have been previously identified for some of the genes, we did not find evidence that a majority of transcripts initiate at the expected distance downstream of these boxes. Our data suggest that differences in the location of transcriptional start sites contribute to the observed complexity in receptor repertoire patterns.

Animals↗
PubMed2000

Novel mouse type D endogenous proviruses and ETn elements share long terminal repeat and internal sequences.

The repetitive ETn (early transposon) family of sequences represents an active "mobile mutagen" in the mouse genome. The presence of long terminal repeats (LTRs) and other diagnostic features indicate that ETns are retrotransposons but they contain no long open reading frames or documented similarity to the genes of known retroviruses or other retroelements. Thus, the mechanisms responsible for the mobility of this family have been unknown. In this study, we used computer searches to detect a small region of previously unrecognized type D retroviral pol homology within ETn elements. This small region was used to isolate two mouse endogenous proviral elements with gag, pro, and pol genes similar to simian type D viruses. This new family of mouse endogenous proviruses, termed MusD, is present in several hundred copies in the genome. Interestingly, the MusD LTRs, 3' internal region, and the 5' region expected to contain the packaging signal are very closely related to members of the ETn subfamily that have recently transposed. Analysis of different mouse strains indicates that MusD elements predate the existence of the mobile subfamily of ETns. These findings indicate that the ETn family was likely created via recombination events resulting in a near complete substitution of MusD coding sequences with unrelated DNA. Furthermore, these results suggest that ETn transcripts retrotranspose using proteins provided by MusD proviruses.

Amino Acid Sequence↗
PubMed1999

Endogenous retroviruses provide the primary polyadenylation signal for two new human genes (HHLA2 and HHLA3).

By screening the expressed sequence tag (EST) database, we identified transcripts of two new human genes that are polyadenylated within a long terminal repeat (LTR) of the HERV-H endogenous retrovirus family. The first gene, termed HHLA2, is represented by two EST clones and one cDNA clone, all of which have a polyadenylated LTR as their 3' end. The gene has an open reading frame (ORF) of 414 amino acids with three immunoglobulin-like domains and is expressed primarily in intestinal tissues, kidney, and lung. Seven small EST clones from several different tissues were found for the second gene, termed HHLA3. As with HHLA2, all HHLA3 ESTs utilized a HERV-H LTR as the polyadenylation signal. Three types of alternatively spliced HHLA3 transcripts that could encode proteins of 76, 121, or 153 amino acids were detected. Interestingly, the ORF for two of these transcripts continues into the LTR. For both HHLA2 and 3, no major human transcripts that utilized a non-LTR polyadenylation signal were detected. Analysis of RNA from baboon, which lacks the LTRs at these genomic loci, showed that the baboon HHLA2 and 3 genes use other polyadenylation signals. This study demonstrates that ancient retroviral insertions have assumed gene regulatory functions during the course of human evolution.

Amino Acid Sequence↗
PubMed1999

Intergenic splicing between a HERV-H endogenous retrovirus and two adjacent human genes.

We previously reported that a long terminal repeat (LTR) of a human endogenous retrovirus of the HERV-H family promotes expression of a cellular fusion transcript in teratocarcinoma cell lines. This transcript was termed PLA2L due to two regions of similarity to the secreted form of phospholipase A2. In this study, evidence is presented indicating that this transcript appears to be the result of intergenic splicing between the HERV-H element and two independent downstream genes. The 5' gene has been named HHLA1 (HERV-H LTR-associating 1) and is of unknown function but shows sequence conservation in other mammals. The 3' gene is now known to encode human otoconin-90 (OC90) which, in mice, is a major protein expressed in the fetal inner ear. Evidence for intergenic splicing of these two genes includes: (1) the isolation of LTR-driven HHLA1 transcripts, unspliced to otoconin-90 exons, with variable sites of polyadenylation; (2) the cloning of both the putative human intergenic genomic region and the novel 5' terminus of the mouse otoconin-90 gene; (3) the identification of homologous potential signal sequences in the 5' region of mouse otoconin-90 and in the middle of the PLA2L transcript; and (4) the lack of detectable chromosomal rearrangements involving this region in teratocarcinoma cells. The PLA2L transcript therefore represents a rare example of intergenic splicing of two closely linked genes. We hypothesize that human HHLA1 and OC90 are normally expressed independently from different promoters but are expressed from the LTR promoter and spliced together in teratocarcinoma cells. It is tempting to speculate that the high activity of the LTR promoter in this cell type may induce transcriptional fusion between these two genes.

5' Untranslated Regions↗
PubMed1998

Role of conserved glycosylation site unique to murine class I MHC in recognition by Ly-49 NK cell receptor.

The recognition of class I MHC molecules on target cells by the Ly-49 family of receptors regulates NK cytotoxicity. Previous studies have suggested that carbohydrates are involved in the recognition of class I MHC by Ly-49, although their precise role remains unclear. Here, we examined the role of asparagine-linked carbohydrates of the murine class I MHC in the binding to Ly-49A and Ly-49C. We have generated H-2Dd mutants that lack the highly conserved glycosylation sites at amino acid residues 86 in the alpha1 domain and 176 in the alpha2 domain, respectively. These mutant Dd cDNAs were transfected into leukemic cell lines, and the binding of the transfected cells to COS cells expressing Ly-49A or Ly-49C, as well as their susceptibility to lysis by Ly-49A+ NK cells, was examined. Only the mutation of the alpha2 domain glycosylation site significantly reduced the binding of Dd to Ly-49A and Ly-49C. Cells expressing Dd with the mutation at this site were partially resistant to killing by Ly-49A+ NK cells. These results suggest that, while carbohydrates linked to residue 176 seem to function as a part of the ligand structure for the Ly-49 family of NK receptors, there are additional structural features involved in this recognition. This glycosylation site is highly conserved among murine class I MHC but is not found among those of other species, suggesting that its role is unique to the murine immune system. It further suggests that murine class I MHC and Ly-49 gene families may have evolved in concert.

Amino Acid Sequence↗
PubMed1998

Localization of five new Ly49 genes, including three closely related to Ly49c.

Nine genes belonging to the mouse Ly49 multigene family of natural killer cell receptors have been identified to date. Two of these genes, Ly49h and i, are very closely related to the well characterized Ly49c gene in the carbohydrate recognition domain. Here we show by Southern blotting that at least two additional new sequences exist in C57BL/6 mice that are also closely related to Ly49c in the carbohydrate recognition domain. Furthermore, in contrast to Ly49a, extensive variation in the arrangement and number of Ly49c-related genes in different mouse strains was observed. To characterize and localize the new Ly49c-related genes in C57BL/6 mice, we isolated and mapped genomic P1 clones hybridizing to an Ly49C exon 7 probe. Locations and the relative order of all Ly49 genes found within the clones was determined. We also used polymerase chain reaction to sequence exons 2, 4, and 7 from all genes. In this manner, we identified five new potential Ly49 genes which have been tentatively termed Ly49j-n. Ly49j, k, and n belong to the Ly49c-related subfamily, whereas Ly49l and Ly49m are most similar to Ly49d and g, respectively. Interestingly, the members of the Ly49c-related subfamily are not clustered as a unit but are interspersed among other Ly49 genes. These results illustrate the complex nature of the Ly49 gene family and should aid in the understanding of functions, such as the mediation of hybrid resistance, in which Ly49c-related genes play a role.

Animals↗
PubMed1997

Genomic structure and evolution of a novel gene (PLA2L) with duplicated phospholipase A2-like domains.

In a previous study, we isolated a novel human cDNA with two domains of homology to secreted phospholipase A2 (sPLA2) embedded within a much larger open reading frame. The corresponding gene, termed PLA2L, is also unusual in that it is transcribed from an endogenous retroviral long terminal repeat promoter in teratocarcinoma cell lines. The associated retroviral element, a member of the HERV-H family of sequences, is found within an intron of the human PLA2L gene and has apparently assumed transcriptional regulatory functions at this locus. In this study we have isolated genomic clones spanning the human PLA2L locus and have determined the intron/exon structure of the PLA2-like domains. This intron/exon structure is very similar to that of known sPLA2s despite the fact that the PLA2L gene is highly diverged and has a novel duplicated structure. We also mapped PLA2L to chromosome 8q24, a location that differs from the known locations of human sPLA2s. Genomic PCR across primate species was performed to determine the approximate time of integration of the HERV-H element. Results indicate that the element integrated 15-20 million years ago since it is present in chimpanzee and gorilla but absent in orangutan and lower primates. Although the function of the PLA2L gene is not known, genomic Southern analyses suggest evolutionary conservation in mammals. These results contribute to our understanding of the unique and complex evolutionary history of the PLA2L gene.

Amino Acid Sequence↗
PubMed1996

Heterogeneity among Ly-49C natural killer (NK) cells: characterization of highly related receptors with differing functions and expression patterns.

Ly-49C is a member of the polymorphic family of murine NK cell inhibitory receptors. The 5E6 antibody that defines a subset of NK cells responsible for the rejection of parental H-2d bone marrow by F1 mice has been shown previously to react with Ly-49C. Here, the 5E6 antibody was found to detect two Ly-49C-related molecules in B6 mice. Two cDNA clones were isolated from B6 NK cells, one identical to previously reported Ly-49CB6 and the other a novel cDNA. The deduced amino acid sequence of the latter differs from that of Ly-49CBALB at only 4 residues, whereas the previously reported Ly-49CB6 differs at 22 residues. Flow cytometric analyses of COS cells transfected with the two cDNAs showed that the 5E6 antibody binds to both Ly-49 molecules, while another anti-Ly-49C antibody, 4LO3311, binds to the newly described Ly-49C but not the previously reported Ly-49CB6. Two-color flow cytometric analysis detected 5E6+4LO3311- as well as 5E6+4LO3311+ subsets of NK cells from B6, but not BALB/c, mice. The level of Ly-49C expression on B6 NK cells detected by the 4LO3311 antibody was substantially lower than that on BALB/c NK cells. Binding specificity of the novel Ly-49CB6 was indistinguishable from that of Ly-49CBALB, whereas no binding was detectable with previously reported Ly-49CB6. These results demonstrate that the newly described Ly-49CB6, not the previously reported Ly-49CB6, is the probable B6 allelic form of Ly-49C. The previously reported Ly-49CB6 must be encoded by a separate gene and should be renamed Ly-49I. The implication of these results with respect to the role of Ly-49C in hybrid resistance is discussed.

Amino Acid Sequence↗
PubMed1996

Transcripts from a novel human KRAB zinc finger gene contain spliced Alu and endogenous retroviral segments.

During the course of an investigation into the potential effects of endogenous retroviruses on adjacent gene expression, we isolated two cDNA clones containing a small sequence segment belonging to the human endogenous retrovirus family, HERV-H. Characterization of the clones revealed that they represent transcripts from a novel KRAB zinc finger gene termed ZNF177. The two cDNA clones differ at their 5' termini and in the presence of a 559-bp internal exon. The clone containing this internal exon has six imperfect zinc finger motifs followed by seven perfect copies of the C2H2 type but has a frame shift between the KRAB domain and the downstream zinc finger region. The smaller clone lacks the six imperfect motifs and has an intact ORF. The 5' putative untranslated regions of both cDNAs contain an 86-bp HERV-H env segment and a segment of an Alu repeat, both in the antisense orientation, that have been incorporated by splicing. RT-PCR experiments show evidence of alternative splicing but the majority of transcripts appear to contain the Alu and env segments. Genomic PCR and hybridization experiments suggest that a partial HERV-H element is integrated within the ZNF177 locus, which Southern analysis has shown to be a single-copy gene. Northern and RT-PCR analyses suggest that ZNF177 is transcribed at a low level in a variety of cell types.

Amino Acid Sequence↗
PubMed1996

Expression of novel-transposon-containing mRNAs in human T cells.

The HERV-H family of endogenous retrovirus like elements is the largest such human family known. Using an HERV-H LTR probe, 6 and 4.5 kb transcripts were detected by Northern blot analysis which were induced in normal peripheral T cells after treatment with phytohaemaglutinin (PHA). Expression was not evident 30 min after treatment with phorbol ester, was increased within 3-4 h after treatment, reached a maximum after 8 h and then declined to low levels 24 h after treatment. Expression was inhibited totally by cycloheximide (10 micromolar) and by the immunosuppressant cyclosporin A (1 microgram/ml). Using probes specific for the U3 and U5 regions of the HERV-H LTR, in combination with internal HERV-H probes, evidence was obtained that the 6 and 4.5 kb transcripts are polyadenylated from an HERV-H LTR. A cDNA library was constructed from T cells which had been treated with PHA for 8 h and a 1.7 kb clone was isolated using the HERV-H LTR probe. The insert contained a novel tandem array of an Alu, a LINE-1 element, two endogenous retroviral LTRs and an A-T-rich sequence. The A-T-rich sequence contained multiple copies of AUUUA mRNA regulatory motifs. Because of its high expression level, defined transcription kinetics, novel cassette-like composition and the presence of conserved mRNA stabilization sequences, we hypothesize that the transcript may play a biological role during T cell activation.

Base Sequence↗
PubMed1995

HERV-H endogenous retroviruses: presence in the New World branch but amplification in the Old World primate lineage.

The evolutionary origin and age of the HERV-H family of human endogenous retrovirus-like sequences was investigated in this study. HERV-H elements exist in approximately 900 partially deleted copies and 50-100 more intact forms in humans and Old World monkeys. However, their possible presence in more divergent species is unknown. We have isolated a 1.6-kb genomic DNA segment from the New World monkey marmoset that had been PCR amplified using human HERV-H primers. DNA and protein comparisons and database searches indicate that this marmoset clone is more closely related to human HERV-H elements than to any other sequence, indicating that HERV-H-related sequences do exist in New World monkeys. In contrast to the high copy numbers of deleted elements in Old World primates. Southern blot analysis shows that such elements are present in less than 50 copies in two different species of New World monkey. To estimate evolutionary ages of the common deleted form of the element, a selected DNA segment from the pol region was compared from multiple human HERV-H elements. This comparison suggests that many HERV-H elements of the abundant deleted subfamily integrated approximately 30-35 million years ago. Very similar percentage divergence values between 5' and 3' long terminal repeats of individual elements of the deleted subfamily also suggest that these elements are close in age. These results indicate that HERV-H elements first appeared in the germline prior to the New World/Old World divergence over 40 million years ago. Interestingly, they remained in low numbers in the New World branch while a subfamily underwent a major amplification in Old World primates before the time of divergence of hominoids from Old World monkeys.

Animals↗
PubMed1995

Spliced HERV-H endogenous retroviral sequences in human genomic DNA: evidence for amplification via retrotransposition.

HERV-H elements are a large family of endogenous retrovirus-like sequences found in approximately 1000 dispersed copies in the genomes of humans and other primates. The most abundant subclass of these elements is a partially deleted form of 5.8 kb which is transcribed primarily as a 5.6-kb unit length RNA and a 3.7-kb spliced derivative. The provirus-like structure of these elements suggests that their numbers have increased in the genome through retrotransposition. However, this has not been demonstrated for HERV-H. To determine if genomic expansion of HERV-H elements involved an RNA intermediate, primate DNAs were screened by PCR for elements that were transcribed, spliced, reverse transcribed, and integrated back into the genome. This PCR screen detected several genomic HERV-H fragments that appear to be derived from spliced transcripts. Interestingly, the presence of one of these fragments is polymorphic in humans, suggesting that its integration was a relatively recent event. Another PCR strategy was used to determine that at least one of the spliced elements has an intact 5' LTR, indicating that it is not simply a "processed pseudogene" or cDNA copy of a HERV-H transcript. Genomic cloning and sequencing of a human locus harboring a spliced element revealed the expected structure, e.g., intact LTRs and flanking 5-bp direct repeats, for a virally retrotransposed element. A genomic library screening method also indicated that very few HERV-H elements (less than 1%) have the structure of processed pseudogenes. These results suggest that most HERV-H elements amplified in the genome as viral retrotransposons.

Animals↗
PubMed1994

Seven patients with respiratory infections due to Corynebacterium pseudodiphtheriticum.

Seven cases of lower respiratory tract infection due to Corynebacterium pseudodiphtheriticum are described. Lower respiratory tract infections with C. pseudodiphtheriticum in immunocompetent patients are usually associated with pre-existing chronic pulmonary disease, and are sometimes associated with endotracheal intubation. Antimicrobial susceptibility testing of these isolates showed uniform sensitivity to penicillin and variable results to erythromycin.

Aged↗
PubMed1994

Management of multisite occupational health services. A hospital/private clinic perspective: changing trends for the 1990s.

A strong regional program can be successful in either a single location or at multiple locations, but it cannot function properly if it is not well organized and staffed properly to deliver services. Extremely important elements to the success of an occupational health clinic that serves a wide geographical region are client retention, a strong information system to manage programs for multiple employers, and cutting-edge leadership that stays in the forefront and educates the business community on emerging issues in occupational health and safety.

Accidents, Occupational↗
PubMed1993

Splicing of a human endogenous retrovirus to a novel phospholipase A2 related gene.

As part of an investigation into the effects of endogenous retroviruses on adjacent genes, we have isolated a cDNA clone derived from the human teratocarcinoma cell line NTera2D1 representing a chimeric transcript in which an endogenous retrovirus-like element, RTVL-H, has been spliced to downstream cellular sequences. The 5' terminus of this clone, termed AF-5, occurs one bp downstream of the predicted transcriptional start site in the RTVL-H long terminal repeat (LTR). AF-5 contains an open reading frame of 689 amino acids beginning within RTVL-H sequences that has two domains of homology with phospholipase A2 (PLA2). These domains, of approximately 120 amino acids each, are 30-38% identical to secreted PLA2s and contain sequence features of both group I and II enzymes. The corresponding AF-5 transcript is 2.5 kb and is derived from a single copy novel gene termed PLA2L. Southern analysis indicates that the RTVL-H element is normally present in human DNA upstream of the PLA2L gene. RTVL-H/PLA2L chimeric transcripts were detected in two independent teratocarcinoma cell lines but not in several other cell lines or primary human tissues. Characterization of additional cDNA clones and PCR analysis indicates that multiple RTVL-H/PLA2L alternatively spliced transcripts are expressed. No evidence has been found for transcription from a non-LTR promoter. These findings strongly suggest that the endogenous LTR promotes expression of the human PLA2L gene in teratocarcinoma cells.

Alternative Splicing↗