Eleven unique cDNA fragments were identified from YAC B30H3, which spans 330 kb in the human majo... more Eleven unique cDNA fragments were identified from YAC B30H3, which spans 330 kb in the human major histocompatibility complex class I region. One fragment (CAT80) was mapped 80 kb telomeric to the HLA-A locus. Using this cDNA fragment as probe, Northern analysis reveals a ubiquitously expressed transcript of about 850 nt in all 16 tissues tested. Based on the cDNA fragment sequence, a full-length cDNA of 858 bp that contains an open reading frame of 378 bp was cloned. Within the putative polypeptide of 126 amino acids, two zinc-ribbon domains were identified: Cx2Cx15Cx2C at the N-terminal and Cx2Cx24Cx2C at the C-terminal. The C-terminal domain is well conserved throughout evolution, including archaea, yeast, Drosophila, nematodes, amphibians, and mammals. The conserved amino acid sequence, CxRCx6Yx3QxRSADEx2TxFxCx2C, is highly homologous to the yeast RNA polymerase A subunit 9 and transcription-associated proteins. Alignment with genomic DNA demonstrates that this gene spans 3.6 kb and consists of four exons and three introns. Crossspecies Northern analysis reveals a mouse homolog of a similar size and with an expression profile similar to those of the human gene. We have named this gene ZNRD1 for zinc ribbon domain-containing 1 protein.
tones, remain largely uncharacterized. We have taken We isolated the human homologue, SUPT5H, of ... more tones, remain largely uncharacterized. We have taken We isolated the human homologue, SUPT5H, of the advantage of the conservation between yeast and mamyeast transcription factor, SPT5. The human homomalian chromatin structural proteins to isolate the logue is 1088 aa long compared to 1063 aa for the yeast mammalian homologues. This approach was successful gene. SUPT5H maps to 19q13, near the ryanodine resince many genes that are essential for cell survival ceptor. Like its family member, SUPT6H, and like are conserved between human and yeast. Based on this yeast SPT5, SUPT5H has a very acidic 5 domain. Like approach, we and others have isolated human homoits family member, SUPT6H, but unlike yeast SPT5 or logues of the yeast SPT4 and SPT6 genes (SUPT4H SPT6, SUPT5H has seven MAP kinase sites at its 5 and SUPT6H, respectively; 3, 4, 6, 10), and we have end. In addition, SUPT5H lacks the novel 6-amino-acid also isolated murine homologues of the yeast SPT4 and
Mice from the MRL strain are prone to develop systemic lupus erythematosus (SLE) and have demonst... more Mice from the MRL strain are prone to develop systemic lupus erythematosus (SLE) and have demonstrated accelerated wound healing and scarless tissue regeneration; however, many of the mechanisms involved in these clinically relevant pathologies are unclear. Prior studies have described macrophage accumulation and functional defects in mice prone to lupus. Monocyte-macrophages have also been shown to have a high degree of plasticity. To determine whether there might be innate differences in the hematopoietic systems of MRL mice, we evaluated hematopoietic progenitor cell content in a variety of tissues and the proliferative responses of derived marrow and thioglycolate (TG)-elicited peritoneal macrophages. Our experiments reveal that MRL mice have significantly lower numbers of circulating blood leukocytes and platelets. Even more strikingly, we found that MRL blood and marrow contain an unusually robust number of unique and assayable macrophage colony-stimulating factor responsive cells which have the characteristics of macrophage colony-forming cell precursors. In culture, in contrast to cells derived from control C57BL/6 mice, this cell type and thioglycolate-elicited peritoneal macrophages from MRL mice can be extensively expanded with just macrophage colony-stimulating factor to acquire an in situ "f-mac-like" (see Y. Zhao, D. Glesne and E. Huberman, A human peripheral blood monocyte-derived subset acts as pluripotent stem cells. Proc. Natl. Acad. Sci. U.S.A. 100, (2003) 2426-2431.) morphology when plated on plastic surfaces. Our results suggest that these increased numbers of macrophage progenitor cells and their potential differentiation plasticity may play a functional role in the onset of systemic lupus erythematosus and may also contribute to the accelerated and scarless tissue regenerative repair response observed in MRL mice.
A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7–2.0 kb were se... more A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7–2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (>20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (≥98% identity), and 16 clones generated nonexact matches (57%–97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the e...
cell differentiation, these initially undifferentiated progenitor cells give rise to the adult re... more cell differentiation, these initially undifferentiated progenitor cells give rise to the adult retina (Adler, 1993; Cepko et al., 1996). As with other developmental systems, this process of retinal development is thought to involve a hierarchy of transcription factors regulating
Expansion of polymorphic CAG and CTG repeats in transcripts is the cause of six inherited neurode... more Expansion of polymorphic CAG and CTG repeats in transcripts is the cause of six inherited neurodegenerative or neuromuscular diseases and may be involved in several other genetic disorders of the central nervous system. To identify new candidate genes, we have undertaken a large-scale screening project for CAG and CTG repeats in human reference cDNAs. We screened 100 128 brain cDNAs by hybridization. We also scanned GenBank expressed sequence tags for the presence of long CAG/CTG repeats in the extremities of cDNAs from several human tissues. Of the selected clones, 286 were found to represent new genes, and 72 have thus far been shown to contain CAG/CTG repeats. Our data indicate that CAG/CTG repeated 10 or more times are more likely to be polymorphic, and that new 3′-directed cDNAs with such repeats are very rare (1/2862). Nine new cDNAs containing polymorphic (observed heterozygote frequency: 0.05-0.90) CAG/CTG repeats have been currently identified in cDNAs. All of the cDNAs have been assigned to chromosomes, and six of them could be mapped with YACs to 1q32-q41, 3p14, 4q28, 3p21 and 12q13.3, 13q13.1-q13.2, and 19q13.43. Three of these clones are highly polymorphic and represent the most likely candidate genes for inherited neurodegenerative diseases and, perhaps, neuropsychiatric disorders of multifactorial origin.
A method that provides standardized data and is relatively inexpensive and capable of high throug... more A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility. In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured. Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.
30 EST/STS have been mapped on human chromosome 19 using a highly specific hncDNA library as a so... more 30 EST/STS have been mapped on human chromosome 19 using a highly specific hncDNA library as a source of transcribed sequences. In addition more than 50 sites constituting 19 families of closely related sequences containing at least one transcribed member each were mapped across the chromosome. Chromosome-19 specific hncDNA clones were hybridized to chromosome 19 cosmids that were previously assembled into contigs covering about 80% of Chr19. The hybridization results were verified by PCR. Such an approach to EST mapping provides information on possible locations of genes as transcribed units of genome and on location of repeated elements used for the priming the hncDNA synthesis. Mapped hncDNA sequences may serve as good starting points for the systematic sequencing of transcribed genomic regions.
Background: A method that provides standardized data and is relatively inexpensive and capable of... more Background: A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on ...
30 EST/STS have been mapped on human chromosome 19 using a highly specific hncDNA library as a so... more 30 EST/STS have been mapped on human chromosome 19 using a highly specific hncDNA library as a source of transcribed sequences. In addition more than 50 sites constituting 19 families of closely related sequences containing at least one transcribed member each were mapped across the chromosome. Chromosome-19 specific hncDNA clones were hybridized to chromosome 19 cosmids that were previously assembled into contigs covering about 80% of Chr19. The hybridization results were verified by PCR. Such an approach to EST mapping provides information on possible locations of genes as transcribed units of genome and on location of repeated elements used for the priming the hncDNA synthesis. Mapped hncDNA sequences may serve as good starting points for the systematic sequencing of transcribed genomic regions.
CAG and CGG expansion is associated with 10 inherited neurological diseases and is thought to be ... more CAG and CGG expansion is associated with 10 inherited neurological diseases and is thought to be involved in other human genetic diseases. To identify new candidate genes, we have undertaken a large-scale screening project for CAG/CTG ([CAG]n) and CGG/GCC ([CGG]n) repeats in human brain reference cDNAs. Here, we present the final classification for 597 cDNAs selected by CAG and CGG hybridization from two libraries (100,128 clones) and the updated characterization of [CAG]n- and [CGG]n-positive cDNAs (repeat polymorphism and cDNA localization). We have selected 124 CAG and 83 CGG hybridization-positive clones representing new genes, from which 49 CAG and 7 CGG repeats could be identified. New [CAG]nand [CGG]nwith more than seven to nine units were rare (1/2000), and perfect [CAG]n9 were more likely polymorphic. Overall, highly polymorphic to monomorphic new [CAG]n> 9 and [CGG]n> 7 were characterized. The comparison of our data with other [CAG]nand [CGG]nresources suggests that ...
Eleven unique cDNA fragments were identified from YAC B30H3, which spans 330 kb in the human majo... more Eleven unique cDNA fragments were identified from YAC B30H3, which spans 330 kb in the human major histocompatibility complex class I region. One fragment (CAT80) was mapped 80 kb telomeric to the HLA-A locus. Using this cDNA fragment as probe, Northern analysis reveals a ubiquitously expressed transcript of about 850 nt in all 16 tissues tested. Based on the cDNA fragment sequence, a full-length cDNA of 858 bp that contains an open reading frame of 378 bp was cloned. Within the putative polypeptide of 126 amino acids, two zinc-ribbon domains were identified: Cx2Cx15Cx2C at the N-terminal and Cx2Cx24Cx2C at the C-terminal. The C-terminal domain is well conserved throughout evolution, including archaea, yeast, Drosophila, nematodes, amphibians, and mammals. The conserved amino acid sequence, CxRCx6Yx3QxRSADEx2TxFxCx2C, is highly homologous to the yeast RNA polymerase A subunit 9 and transcription-associated proteins. Alignment with genomic DNA demonstrates that this gene spans 3.6 kb and consists of four exons and three introns. Crossspecies Northern analysis reveals a mouse homolog of a similar size and with an expression profile similar to those of the human gene. We have named this gene ZNRD1 for zinc ribbon domain-containing 1 protein.
tones, remain largely uncharacterized. We have taken We isolated the human homologue, SUPT5H, of ... more tones, remain largely uncharacterized. We have taken We isolated the human homologue, SUPT5H, of the advantage of the conservation between yeast and mamyeast transcription factor, SPT5. The human homomalian chromatin structural proteins to isolate the logue is 1088 aa long compared to 1063 aa for the yeast mammalian homologues. This approach was successful gene. SUPT5H maps to 19q13, near the ryanodine resince many genes that are essential for cell survival ceptor. Like its family member, SUPT6H, and like are conserved between human and yeast. Based on this yeast SPT5, SUPT5H has a very acidic 5 domain. Like approach, we and others have isolated human homoits family member, SUPT6H, but unlike yeast SPT5 or logues of the yeast SPT4 and SPT6 genes (SUPT4H SPT6, SUPT5H has seven MAP kinase sites at its 5 and SUPT6H, respectively; 3, 4, 6, 10), and we have end. In addition, SUPT5H lacks the novel 6-amino-acid also isolated murine homologues of the yeast SPT4 and
Mice from the MRL strain are prone to develop systemic lupus erythematosus (SLE) and have demonst... more Mice from the MRL strain are prone to develop systemic lupus erythematosus (SLE) and have demonstrated accelerated wound healing and scarless tissue regeneration; however, many of the mechanisms involved in these clinically relevant pathologies are unclear. Prior studies have described macrophage accumulation and functional defects in mice prone to lupus. Monocyte-macrophages have also been shown to have a high degree of plasticity. To determine whether there might be innate differences in the hematopoietic systems of MRL mice, we evaluated hematopoietic progenitor cell content in a variety of tissues and the proliferative responses of derived marrow and thioglycolate (TG)-elicited peritoneal macrophages. Our experiments reveal that MRL mice have significantly lower numbers of circulating blood leukocytes and platelets. Even more strikingly, we found that MRL blood and marrow contain an unusually robust number of unique and assayable macrophage colony-stimulating factor responsive cells which have the characteristics of macrophage colony-forming cell precursors. In culture, in contrast to cells derived from control C57BL/6 mice, this cell type and thioglycolate-elicited peritoneal macrophages from MRL mice can be extensively expanded with just macrophage colony-stimulating factor to acquire an in situ "f-mac-like" (see Y. Zhao, D. Glesne and E. Huberman, A human peripheral blood monocyte-derived subset acts as pluripotent stem cells. Proc. Natl. Acad. Sci. U.S.A. 100, (2003) 2426-2431.) morphology when plated on plastic surfaces. Our results suggest that these increased numbers of macrophage progenitor cells and their potential differentiation plasticity may play a functional role in the onset of systemic lupus erythematosus and may also contribute to the accelerated and scarless tissue regenerative repair response observed in MRL mice.
A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7–2.0 kb were se... more A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7–2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (>20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (≥98% identity), and 16 clones generated nonexact matches (57%–97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the e...
cell differentiation, these initially undifferentiated progenitor cells give rise to the adult re... more cell differentiation, these initially undifferentiated progenitor cells give rise to the adult retina (Adler, 1993; Cepko et al., 1996). As with other developmental systems, this process of retinal development is thought to involve a hierarchy of transcription factors regulating
Expansion of polymorphic CAG and CTG repeats in transcripts is the cause of six inherited neurode... more Expansion of polymorphic CAG and CTG repeats in transcripts is the cause of six inherited neurodegenerative or neuromuscular diseases and may be involved in several other genetic disorders of the central nervous system. To identify new candidate genes, we have undertaken a large-scale screening project for CAG and CTG repeats in human reference cDNAs. We screened 100 128 brain cDNAs by hybridization. We also scanned GenBank expressed sequence tags for the presence of long CAG/CTG repeats in the extremities of cDNAs from several human tissues. Of the selected clones, 286 were found to represent new genes, and 72 have thus far been shown to contain CAG/CTG repeats. Our data indicate that CAG/CTG repeated 10 or more times are more likely to be polymorphic, and that new 3′-directed cDNAs with such repeats are very rare (1/2862). Nine new cDNAs containing polymorphic (observed heterozygote frequency: 0.05-0.90) CAG/CTG repeats have been currently identified in cDNAs. All of the cDNAs have been assigned to chromosomes, and six of them could be mapped with YACs to 1q32-q41, 3p14, 4q28, 3p21 and 12q13.3, 13q13.1-q13.2, and 19q13.43. Three of these clones are highly polymorphic and represent the most likely candidate genes for inherited neurodegenerative diseases and, perhaps, neuropsychiatric disorders of multifactorial origin.
A method that provides standardized data and is relatively inexpensive and capable of high throug... more A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility. In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured. Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.
30 EST/STS have been mapped on human chromosome 19 using a highly specific hncDNA library as a so... more 30 EST/STS have been mapped on human chromosome 19 using a highly specific hncDNA library as a source of transcribed sequences. In addition more than 50 sites constituting 19 families of closely related sequences containing at least one transcribed member each were mapped across the chromosome. Chromosome-19 specific hncDNA clones were hybridized to chromosome 19 cosmids that were previously assembled into contigs covering about 80% of Chr19. The hybridization results were verified by PCR. Such an approach to EST mapping provides information on possible locations of genes as transcribed units of genome and on location of repeated elements used for the priming the hncDNA synthesis. Mapped hncDNA sequences may serve as good starting points for the systematic sequencing of transcribed genomic regions.
Background: A method that provides standardized data and is relatively inexpensive and capable of... more Background: A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on ...
30 EST/STS have been mapped on human chromosome 19 using a highly specific hncDNA library as a so... more 30 EST/STS have been mapped on human chromosome 19 using a highly specific hncDNA library as a source of transcribed sequences. In addition more than 50 sites constituting 19 families of closely related sequences containing at least one transcribed member each were mapped across the chromosome. Chromosome-19 specific hncDNA clones were hybridized to chromosome 19 cosmids that were previously assembled into contigs covering about 80% of Chr19. The hybridization results were verified by PCR. Such an approach to EST mapping provides information on possible locations of genes as transcribed units of genome and on location of repeated elements used for the priming the hncDNA synthesis. Mapped hncDNA sequences may serve as good starting points for the systematic sequencing of transcribed genomic regions.
CAG and CGG expansion is associated with 10 inherited neurological diseases and is thought to be ... more CAG and CGG expansion is associated with 10 inherited neurological diseases and is thought to be involved in other human genetic diseases. To identify new candidate genes, we have undertaken a large-scale screening project for CAG/CTG ([CAG]n) and CGG/GCC ([CGG]n) repeats in human brain reference cDNAs. Here, we present the final classification for 597 cDNAs selected by CAG and CGG hybridization from two libraries (100,128 clones) and the updated characterization of [CAG]n- and [CGG]n-positive cDNAs (repeat polymorphism and cDNA localization). We have selected 124 CAG and 83 CGG hybridization-positive clones representing new genes, from which 49 CAG and 7 CGG repeats could be identified. New [CAG]nand [CGG]nwith more than seven to nine units were rare (1/2000), and perfect [CAG]n9 were more likely polymorphic. Overall, highly polymorphic to monomorphic new [CAG]n> 9 and [CGG]n> 7 were characterized. The comparison of our data with other [CAG]nand [CGG]nresources suggests that ...
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Papers by G. Lennon