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T S Okada

Publications and source records attributed to T S Okada.

At least 37 records · Page 2Linked to original sources
PubMed1987

Lens-specific enhancer in the third intron regulates expression of the chicken delta 1-crystallin gene.

We have previously shown that the tissue specificity determinant of the chicken delta 1-crystallin gene lies 3' of position -100 (Hayashi et al. 1985). Since the promoter of the gene (delta 1-crystallin promoter) did not show any tissue specificity, we examined various segments of the delta 1-crystallin gene for a tissue-specific enhancer activity by placing each segment downstream of a heterologous transcriptional unit coding for chloramphenicol acetyltransferase (CAT) and by transfecting chicken tissues in primary culture. We found that a segment spanning the third intron bears a strong lens-specific enhancer activity. This "delta 1-crystallin enhancer" activates transcription from the delta 1-crystallin promoter 20- to 40-fold in lens cells and to various degrees with other promoters. Deletion analysis of the enhancer region indicated that it covered nearly 1 kb but did not indicate clear-cut boundaries. For its enhancer effect the core region of 120 bp and associations with certain adjoining regions were required. Removal of the enhancer from the gene totally abolished delta 1-crystallin expression, and reinsertion of the enhancer in either upstream, internal, or downstream positions restored expression. We conclude that the delta 1-crystallin enhancer is an essential and major determinant for lens-specificity of delta 1-crystallin expression.

Animals↗
PubMed1986

Regulation of delta-crystallin expression: an investigation by transfer of the chicken gene into mouse cells.

In order to understand how crystallin genes are regulated so that they would be expressed with a high lens specificity, we introduced a cloned delta-crystallin gene of the chicken and its derivatives into various mouse cells. In such a heterologous combination of the gene and the recipient cells, chicken delta-crystallin was expressed in lens cells with a high preference over other cell types. The high expression in the lens is basically ascribed to interactions between the putative lens-specific mechanisms for transcription and the base pairs from -80 to +35 encompassing the transcriptional initiation site of the delta-crystallin gene. A non-specific low expression was also observed. This may account for ectopic delta-crystallin expression which takes place in certain non-lenticular embryonic tissues of the chicken.

Animals↗
PubMed1986

Transient expression of a 'lens-specific' gene, delta-crystallin, in the embryonic chicken adenohypophysis.

A characteristic protein of the lens, delta-crystallin, has been reported previously to be present in the embryonic chicken adenohypophysis. We confirmed this earlier finding by biochemical detection of delta-crystallin protein using a monoclonal antibody and delta-crystallin mRNA using a specific cDNA probe. We estimate the concentration of delta-crystallin and its mRNA in the 3.5-day embryonic chicken adenohypophysis to be approximately 1/3,000 and 1/5,000 of the respective value found in lens. Tissue culture revealed that cells positive for delta-crystallin comprise about 30% of embryonic adenohypophysis and are randomly scattered in this organ. No lentoid formation was observed during the culture period.

Animals↗
PubMed1986

Production of transgenic fish: introduction and expression of chicken delta-crystallin gene in medaka embryos.

To produce a model of transgenic fish, recombinant plasmids containing chicken delta-crystallin gene were microinjected into the oocyte nucleus of a small teleost, medaka (Oryzias latipes). About 50% of the microinjected oocytes developed to 7-day-old embryos. By Southern blotting delta-crystallin gene was detected in 4 of 8 embryos, and, by Western blotting, delta-crystallin polypeptides in 5 of 16. In 1 of 6 examined histologically, delta-crystallin DNA was detected in all the tissues, and delta-crystallin polypeptides, in many of the tissues including the lens. Thus, the exogenous gene and/or its products were detected in 10 of 30 embryos examined. This is the first report of successful production of transgenic fish.

Animals↗
PubMed1985

Nucleotide sequence of a chicken delta-crystallin gene.

We have determined the complete nucleotide sequence of one of the two non-allelic delta-crystallin genes in the chicken, arbitrarily designated delta-gene 1, using a genomic clone (lambda g delta 106) containing the entire gene sequence. By comparison of the genomic sequence and the delta-crystallin cDNA sequence previously determined, we have identified exon sequences in the genomic sequence. Thus, the presence of 17 exons and 16 introns in the gene has been clarified. The delta-crystallin polypeptide deduced from the exon sequences consists of 465 amino acids which is larger, by 19 amino acid residues, than the polypeptide deduced from the cDNA sequence previously reported. Re-examination of the cDNA sequence using the same cDNA clone previously used shows that the present exon sequences are correct and the molecular weight of the deduced delta-crystallin polypeptide is 50,615 daltons instead of the previously reported value of 48,447 daltons. In addition, some structural features of the delta-crystallin gene including putative expression signals are discussed.

Alleles↗
PubMed1985

Tissue-specific regulation of a chicken delta-crystallin gene in mouse cells: involvement of the 5' end region.

A cloned delta-crystallin gene of the chicken is preferentially expressed in lens cells after introduction into various mouse tissues. The level of expression in the lens epithelium is 20 times higher than in fibroblasts. Taking advantage of this system, we attempted to define regulatory regions of the delta-crystallin gene using a variety of deletion and substitution mutants. The results indicate that tissue-specific regulation of the delta-crystallin gene is mediated by the 5' end region of the gene; sequences upstream from -93 are not required for expression and sequences downstream from +58 are not involved in tissue specificity. The high expression in lens cells requires 5' flanking sequences of 80-bp long from the cap site, whereas the low expression in fibroblasts requires an additional 12 bp upstream sequence. Expression of both types is lost in a mutant with only 51 bp of the 5' flanking sequence. Thus, fine deletion analysis demonstrated that expression in lens cells and expression in fibroblasts are distinct not only in level but in regulation.

Amino Acid Sequence↗
PubMed1985

DNA sequences responsible for tissue-specific expression of a chicken alpha-crystallin gene in mouse lens cells.

We have studied the DNA sequences required for high-level expression of a cloned chicken alpha-crystallin gene by introducing a hybrid alpha/delta-crystallin gene into nuclei of mouse lens epithelial cells in primary culture. The level of transient expression of the hybrid gene consisting of the 5' upstream promoter region of the alpha-crystallin gene fused to the structural portion of the delta-crystallin gene was determined by Western blot analysis using anti-delta-crystallin serum. The hybrid gene appears to be expressed in a tissue-specific manner, since it is active in mouse lens cells but not in fibroblasts or in L cells. The DNA sequences located 242-189 bp upstream from the transcription initiation site are required for high-level expression in lens cells. They are active when their orientation is reversed at the original site or when placed approximately 1.7 kbp downstream from the cap site in the second intron of the hybrid gene in either orientation. When these DNA sequences were replaced by the enhancer sequences of Moloney murine leukemia virus, the hybrid gene was expressed in both lens cells and fibroblasts.

Animals↗
PubMed1985

Oculopotency of embryonic quail pineals as revealed by cell culture studies.

Pineal bodies from 8-day-old quail embryos were dissociated and cultured in order to examine their potency for differentiation under in vitro conditions. Polygonal pigment cells and lentoid bodies started to differentiate after about 2 and 4 weeks, respectively. Lentoid bodies were shown immunologically to contain all classes of crystallins. The results indicate that embryonic pineal cells of avian species retain 'oculopotency' to differentiate into several types of ocular cells.

Animals↗
PubMed1985

A monoclonal antibody disrupting calcium-dependent cell-cell adhesion of brain tissues: possible role of its target antigen in animal pattern formation.

The Ca2+-dependent cell-cell adhesion system (CDS) is thought to be essential for the formation and maintenance of cell adhesion in a wide variety of tissues. Previous studies suggested that CDS has some cell-type specificity; for example, the monoclonal antibody ECCD-1 selectively recognizes CDS of certain epithelial tissues in mouse embryos but not nervous tissues. In the present study, we have obtained a monoclonal antibody, designated NCD-1, that disrupts connections between brain cells of mouse embryos. A series of experiments suggested that NCD-1 specifically recognizes CDS. We then determined the distribution of the NCD-1 antigen in various mouse tissues. NCD-1 reacted with cells of the following tissues and cell lines: nervous tissues from various sources, lens, striated muscle, cardiac muscle, glioma G26-20, adrenocortical tumor Y1, and melanoma B16. None of these cells reacted with ECCD-1, and the cells reactive with ECCD-1 did not react with NCD-1. There was also a class of cells that did not react with either ECCD-1 or NCD-1. These results suggest that cells in the body can be classified into at least three groups containing CDS of differing specificities. A map of the tissue localization of these different classes of CDS also suggests that the expression of cell-type-specific cell adhesion molecules in each tissue plays a crucial role in adhesion between the same cell types and segregation of different cell types in processes essential for animal morphogenesis.

Animals↗
PubMed1984

The nucleotide sequence of a complete chicken delta-crystallin cDNA.

The nucleotide sequence of a full length cDNA of delta-crystallin mRNA from chicken lens has been determined using a delta-crystallin cDNA clone (pB delta 11), which represents the mRNA sequence of 1530 nucleotides from the poly(A) junction but does not contain the 5'-terminal sequence of 44 nucleotides of the mRNA. The 5'-terminal sequence of the mRNA, absent in the cDNA clone, has been determined with a stretch of cDNA sequence by the primer extension procedure. The amino acid sequence deduced from the nucleotide sequence is consistent with the amino acid sequences of several tryptic peptides, the total amino acid composition, and the mol. wt. of delta-crystallin estimated by SDS-polyacrylamide gel electrophoresis. The computer-assisted analysis predicts high alpha-helical content throughout the polypeptide. Sequence analyses have revealed that gene 1 encodes the mRNA from which the cDNA clone was derived.

Amino Acid Sequence↗
PubMed1984

Differentiation-dependent expression of the chicken delta-crystallin gene introduced into mouse teratocarcinoma stem cells.

PCC3 mouse teratocarcinoma (TCC) stem cells were cotransfected with either the plasmid p delta C-1A or p delta C-1B carrying the chicken delta-crystallin gene, and with the plasmid pSV2gpt containing the selectable bacterial xanthine-guanine phosphoribosyltransferase (XGPRT) gene, using the calcium phosphate technique. Nine transformed PCC3 stem cell lines, each of which was clonally derived from respective colonies surviving after the selection process, were isolated. Southern blot analysis revealed that all of them stably maintained delta-crystallin sequences associated with high mol. wt. cellular DNA after propagation in non-selective medium in vitro, and after the production of solid tumors in the syngenic host mice. Six cell lines contain the intact delta-crystallin gene sequence and eight contain the gpt sequence. The number of delta-crystallin DNA copies was highly variable among transformed lines, 1-500 delta-crystallin genes per diploid mouse genome. No expression of the exogenous genes was detected in the transformed cells as long as they were in the undifferentiated state. However, the synthesis of delta-crystallin in certain types of cells was detected immunohistologically in three lines after the differentiation. The positive cell types were unique to each line, skeletal muscle in Y delta-9, certain columnar epithelia in Y delta-2, and unidentified spindle-shaped cells in Y delta-3. Authentic delta-crystallin polypeptides with a mol. wt. of 48 000 are synthesized upon differentiation of line Y delta-3 in solid tumors in syngenic mice.

Animals↗
PubMed1983

Tissue-specific expression of a cloned chick delta-crystallin gene in mouse cells.

Crystallins are a group of soluble proteins specific to vertebrate lenses, and have been used successfully as molecular markers for studying lens differentiation. The synthesis and accumulation of delta-crystallin in the differentiating lens cells of chick, in particular, have been extensively studied (for review see refs 1-3). Recently, we have cloned a continuous stretch of a chick delta-crystallin gene which is specifically expressed in lens cells. We have injected this gene into nuclei of various mouse somatic cells. delta-Crystallin is totally absent from mammalian lenses in which it is replaced by gamma-crystallin, thus this xenogeneic injection system should allow us to study tissue-specific gene expression. We report here experiments which demonstrate that (1) the cloned delta-crystallin gene of chick is expressed in the mouse lens epithelial cells as efficiently as in the homologous chick cells; (2) delta-crystallin synthesized in the mouse lens epithelium has the native molecular weight, indicating correct splicing of the gene transcripts; (3) the expression is inefficient in the non-lens tissues examined, suggesting lens-specific expression of the delta-crystallin gene in the xenogenic environment. To our knowledge, this is the first demonstration that a cloned gene shows preferential expression in homologous cell types of different species.

Animals↗
PubMed1983

Gene coding for a lens-specific protein, delta-crystallin, is transcribed in nonlens tissues of chicken embryos.

The question of whether or not delta-crystallin gene (delta-gene) is transcribed in tissues of chicken embryos other than lens was examined by Northern blot analysis, using cloned delta-crystallin cDNA as a probe. delta-Crystallin mRNA was detected in neural retinas, brains, and limb buds of 3.5-day-old embryos. However, the distribution of delta-gene transcripts is limited to lens only in posthatched chickens. It was indicated that the ratio of large delta-crystallin RNA, probably precursor RNA, to mature delta-crystallin mRNA is always much higher in nonlens tissues.

Animals↗
PubMed1983

The accumulation of delta-crystallin mRNA in transdetermination and transdifferentiation of neural retina cells into lens.

By means of hybridization with DNA complementary to delta-crystallin mRNA (delta-mRNA) sequences (delta-cDNA), the levels of delta-mRNA in three different culture systems of 8-day-old chick embryonic neural retina were determined. After 30 days of culturing in vitro, the level of delta-mRNA in cells cultured under conditions of spreading cultures (SpC) was 50 times higher than in the cells maintained in aggregate cultures (AgC) throughout. When the cells pre-cultivated in SpC for an initial 10 days were transferred into AgC, the delta-mRNA level in 30-day cultures was 40 times higher than that in 3-day SpC. The level of delta-mRNA in neural retina in situ was negligible, but it became detectable in 10-day SpC. The initial appearance of detectable delta-mRNA in 10-day SpC coincides with the timing of 'transdetermination' of neural retina cells into lens cells.

Animals↗