Joan Krakowsky | Keiser University (original) (raw)

Papers by Joan Krakowsky

Research paper thumbnail of Promoteurs de l'exon 1 et de l'exon 3 du facteur humain de croissance du tissu nerveux

L'invention concerne un nouveau promoteur de l'exon 1 du facteur humain de croissance du ... more L'invention concerne un nouveau promoteur de l'exon 1 du facteur humain de croissance du tissu nerveux, un promoteur de l'exon 3 de ce facteur, des fragments de ce promoteur ainsi que des formes modifiees de celui-ci. L'invention concerne egalement des vecteurs contenant de tels promoteurs, des cellules transformees a l'aide de ceux-ci, notamment des modeles animaux et des animaux transgeniques contenant une telle sequence, ainsi que des procedes de dosage effectues au moyen de ces promoteurs.

Research paper thumbnail of Human nerve growth factor exon 1 and exon 3 promoters

Research paper thumbnail of Human gamma- to beta-globin gene switching using a mini construct in transgenic mice

Molecular and Cellular Biology, 1992

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) ... more The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down...

Research paper thumbnail of Ocular pathology in mice with a transgenic insertion at the microphthalmia locus

Journal of submicroscopic cytology and pathology, 1993

A DNA insertional mutation at the microphthalmia locus (mi) in a transgenic mouse has been develo... more A DNA insertional mutation at the microphthalmia locus (mi) in a transgenic mouse has been developed and given the allele symbol of mitg (Krakowsky et al., 1993). Mice homozygous for this transgene have eyes markedly reduced in size and relatively unpigmented. In this study, we examined the morphology of these eyes using light and electron microscopy. Transgenic homozygous (mitg/mitg) animals have a structurally normal choroid which lacks melanocytes but contains occasional leukocytes. The elastica of Bruch's membrane is absent except in an occasional site. The retinal pigmented epithelium (RPE) appears dramatically abnormal. It displays cellular heterogeneity, residual basal infoldings and apical microvilli, rare and immature melanosomes, and numerous cilia-like structures. Occasionally, cells of the RPE appear to have extruded into or from the choroid. The photoreceptor cells are devoid completely of outer segments and partially of inner segments. Numerous active macrophages a...

Research paper thumbnail of A DNA insertional mutation results in microphthalmia in transgenic mice

Transgenic Research, 1993

Transgenic mice were produced by microinjection of a human Ay-globin gene construct containing si... more Transgenic mice were produced by microinjection of a human Ay-globin gene construct containing site 2 of the locus control region and the gy-globin gene with its 3' enhancer sequence. One transgenic mouse line (5'HS2yen91) displayed an altered phenotype when the insertion event of this transgenic line was homozygous. These animals lack the normal pigmentation seen in their hemizygous and non-transgenic littermates, thus appearing white with unpigmented eyes. In addition, their eyes are underdeveloped, consistent with the phenotype associated with mutations at the microphthalmia (mi) locus. Backcrosses of transgenic mice with mi mutant mice result in phenotypes showing a lack of complementation, demonstrating that the site of transgene insertion is allelic with mi. Electron microscopic analysis of hair follicles and culturing of melanocytes from the skin of transgenic animals reveals an absence of cutaneous melanocytes in homozygotes and aberrant growth and morphology of the melanocytes isolated from hemizygous animals. The results presented here summarize the effects of this new allele of the mi locus.

Research paper thumbnail of Analysis of Possible Repressor Elements in the 5′-Flanking Region of the Human β-Globin Gene

DNA, 1989

Human beta-globin gene expression is confined predominantly to the adult with little or no expres... more Human beta-globin gene expression is confined predominantly to the adult with little or no expression of this gene occurring during embryonic or fetal life. The lack of expression of this gene in embryonic and fetal erythroid tissue could be due to the absence of required positive regulatory factors in these cells or the presence of negative regulatory factors which prevent expression of the adult globin gene. To test the repressor model, we have used a gel electrophoretic mobility shift assay to identify regions in the human beta-globin gene which bind proteins found in K562 cells, a cell line that expresses embryonic and fetal globins but not adult beta-globin. DNA fragments comprising the entire human beta-globin gene were assayed using nuclear proteins from K562 cells, and four regions were found that bind proteins. These are located within the 5'-flanking region, within the first and second introns, and at the 3'-flanking region of the gene. Previous studies have suggested the presence of potential repressor sites 5' of exon 2. For this reason, we examined whether the lack of the binding regions in the 5'-flanking sequence allow expression of the human beta-globin gene in transgenic mice during embryonic life. beta-globin gene expression was confined to adult life, indicating that if a transcriptional repressor is responsible for inactivating this gene in embryonic tissue, it is not regulated solely by sequences upstream from -122 bp in the 5'-flanking region of the human beta-globin gene.

Research paper thumbnail of A helix-loop-helix transcription factor-like gene is located at the mi locus

Journal of Biological Chemistry, 1993

The mouse microphthalmia phenotype is complex and consists of one or more of the following phenot... more The mouse microphthalmia phenotype is complex and consists of one or more of the following phenotypic alterations: a lack of pigmentation, small eyes, a mast cell defect, and bone abnormalities. The locus for this allele has been assigned to chromosome 6. A single gene defect that produces such a pleiotropic effect has suggested some involvement at a control point in development. Recently a mutant line of mice carrying a transgene insertion, which represents a new allelic form of mi, was described. The integration site of the transgene from these mi(tg) mice was cloned and analyzed. An exon sequence was discovered adjacent to the insertion. Computer analysis of this nucleotide sequence revealed the presence of a motif indicative of the helix-loop-helix class of transcription factors. The gene was expressed in a number of tissues from wild type animals but was absent in the tissue RNA from mi(tg) mice. Southern blot analysis demonstrated a deletion of some of the genetic material for this gene in the mi(tg) mice. This is consistent with the lack of expression in the mi(tg) mice. Interestingly, when DNA from other mi allelic variants was subjected to a similar analysis, a deletion was also observed in this gene in two other mi lines. Taken together, these data suggest that the gene encoding this new helix-loop-helix DNA-binding protein, and residing in the mi locus, is a strong candidate for the mi gene.

Research paper thumbnail of Promoteurs de l'exon 1 et de l'exon 3 du facteur humain de croissance du tissu nerveux

L'invention concerne un nouveau promoteur de l'exon 1 du facteur humain de croissance du ... more L'invention concerne un nouveau promoteur de l'exon 1 du facteur humain de croissance du tissu nerveux, un promoteur de l'exon 3 de ce facteur, des fragments de ce promoteur ainsi que des formes modifiees de celui-ci. L'invention concerne egalement des vecteurs contenant de tels promoteurs, des cellules transformees a l'aide de ceux-ci, notamment des modeles animaux et des animaux transgeniques contenant une telle sequence, ainsi que des procedes de dosage effectues au moyen de ces promoteurs.

Research paper thumbnail of Human nerve growth factor exon 1 and exon 3 promoters

Research paper thumbnail of Human gamma- to beta-globin gene switching using a mini construct in transgenic mice

Molecular and Cellular Biology, 1992

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) ... more The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down...

Research paper thumbnail of Ocular pathology in mice with a transgenic insertion at the microphthalmia locus

Journal of submicroscopic cytology and pathology, 1993

A DNA insertional mutation at the microphthalmia locus (mi) in a transgenic mouse has been develo... more A DNA insertional mutation at the microphthalmia locus (mi) in a transgenic mouse has been developed and given the allele symbol of mitg (Krakowsky et al., 1993). Mice homozygous for this transgene have eyes markedly reduced in size and relatively unpigmented. In this study, we examined the morphology of these eyes using light and electron microscopy. Transgenic homozygous (mitg/mitg) animals have a structurally normal choroid which lacks melanocytes but contains occasional leukocytes. The elastica of Bruch's membrane is absent except in an occasional site. The retinal pigmented epithelium (RPE) appears dramatically abnormal. It displays cellular heterogeneity, residual basal infoldings and apical microvilli, rare and immature melanosomes, and numerous cilia-like structures. Occasionally, cells of the RPE appear to have extruded into or from the choroid. The photoreceptor cells are devoid completely of outer segments and partially of inner segments. Numerous active macrophages a...

Research paper thumbnail of A DNA insertional mutation results in microphthalmia in transgenic mice

Transgenic Research, 1993

Transgenic mice were produced by microinjection of a human Ay-globin gene construct containing si... more Transgenic mice were produced by microinjection of a human Ay-globin gene construct containing site 2 of the locus control region and the gy-globin gene with its 3' enhancer sequence. One transgenic mouse line (5'HS2yen91) displayed an altered phenotype when the insertion event of this transgenic line was homozygous. These animals lack the normal pigmentation seen in their hemizygous and non-transgenic littermates, thus appearing white with unpigmented eyes. In addition, their eyes are underdeveloped, consistent with the phenotype associated with mutations at the microphthalmia (mi) locus. Backcrosses of transgenic mice with mi mutant mice result in phenotypes showing a lack of complementation, demonstrating that the site of transgene insertion is allelic with mi. Electron microscopic analysis of hair follicles and culturing of melanocytes from the skin of transgenic animals reveals an absence of cutaneous melanocytes in homozygotes and aberrant growth and morphology of the melanocytes isolated from hemizygous animals. The results presented here summarize the effects of this new allele of the mi locus.

Research paper thumbnail of Analysis of Possible Repressor Elements in the 5′-Flanking Region of the Human β-Globin Gene

DNA, 1989

Human beta-globin gene expression is confined predominantly to the adult with little or no expres... more Human beta-globin gene expression is confined predominantly to the adult with little or no expression of this gene occurring during embryonic or fetal life. The lack of expression of this gene in embryonic and fetal erythroid tissue could be due to the absence of required positive regulatory factors in these cells or the presence of negative regulatory factors which prevent expression of the adult globin gene. To test the repressor model, we have used a gel electrophoretic mobility shift assay to identify regions in the human beta-globin gene which bind proteins found in K562 cells, a cell line that expresses embryonic and fetal globins but not adult beta-globin. DNA fragments comprising the entire human beta-globin gene were assayed using nuclear proteins from K562 cells, and four regions were found that bind proteins. These are located within the 5'-flanking region, within the first and second introns, and at the 3'-flanking region of the gene. Previous studies have suggested the presence of potential repressor sites 5' of exon 2. For this reason, we examined whether the lack of the binding regions in the 5'-flanking sequence allow expression of the human beta-globin gene in transgenic mice during embryonic life. beta-globin gene expression was confined to adult life, indicating that if a transcriptional repressor is responsible for inactivating this gene in embryonic tissue, it is not regulated solely by sequences upstream from -122 bp in the 5'-flanking region of the human beta-globin gene.

Research paper thumbnail of A helix-loop-helix transcription factor-like gene is located at the mi locus

Journal of Biological Chemistry, 1993

The mouse microphthalmia phenotype is complex and consists of one or more of the following phenot... more The mouse microphthalmia phenotype is complex and consists of one or more of the following phenotypic alterations: a lack of pigmentation, small eyes, a mast cell defect, and bone abnormalities. The locus for this allele has been assigned to chromosome 6. A single gene defect that produces such a pleiotropic effect has suggested some involvement at a control point in development. Recently a mutant line of mice carrying a transgene insertion, which represents a new allelic form of mi, was described. The integration site of the transgene from these mi(tg) mice was cloned and analyzed. An exon sequence was discovered adjacent to the insertion. Computer analysis of this nucleotide sequence revealed the presence of a motif indicative of the helix-loop-helix class of transcription factors. The gene was expressed in a number of tissues from wild type animals but was absent in the tissue RNA from mi(tg) mice. Southern blot analysis demonstrated a deletion of some of the genetic material for this gene in the mi(tg) mice. This is consistent with the lack of expression in the mi(tg) mice. Interestingly, when DNA from other mi allelic variants was subjected to a similar analysis, a deletion was also observed in this gene in two other mi lines. Taken together, these data suggest that the gene encoding this new helix-loop-helix DNA-binding protein, and residing in the mi locus, is a strong candidate for the mi gene.