Lens-specific expression of the chloramphenicol acetyltransferase gene promoted by 5' flanking sequences of the murine alpha A-crystallin gene in explanted chicken lens epithelia.

Chepelinsky, Ana B.; King, Charles R.; Zelenka, Peggy S.; Piatigorsky, Joram

doi:10.1073/pnas.82.8.2334

Cited by 119 publications

(89 citation statements)

References 42 publications

(32 reference statements)

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“…In fact, wellcharacterized GC box-dependent genes have been either viral or housekeeping cellular genes (e.g., the simian virus 40, herpes simplex virus tk, and dehydrofolate reductase genes), and this is the first example of the involvement of a GC box in the expression of a tissue-specific gene. Among the crystallin genes which have been cloned and whose expression has been characterized (2,4,9,14,19), only the b-crystallin genes have GC boxes in their promoters. It is possible that interactions of a tissue-specific factor(s) with the B-crystallin promoter give rise to a potential for lensspecific b-crystallin expression, and binding of an Spl-like factor to the GC box may activate this expression.…”

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confidence: 99%

In vivo competition of delta-crystallin gene expression by DNA fragments containing a GC box.

Hayashi¹,

Kondoh²

1986

Mol. Cell. Biol.

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Expression of the chicken 8-crystallin gene 1 injected into the nuclei of mouse cells is lens specific. Coinjection of GC box-containing DNA fragments from 8-crystallin, simian virus 40 early, and herpes simplex virus type 1 tk promoters effectively suppressed 8-crystallin expression in the lens, but coinjection with DNA fragments not containing the GC box did not. This suppression was likely due to the competition of an Spl-like transcription factor(s) and indicates involvement of the apparently ubiquitous factor(s) in the tissue-specific expression of the 8-crystallin gene.B-Crystallin is the most abundantly produced lens-specific protein of avians. The cloned 8-crystallin gene 1 (17,18), the active entity of two nonallelic genes of chickens (2,16,23), is expressed with a high lens specificity after nuclear injection into mouse cells (9,11,13). By introducing a series of deletions and substitutions in the gene and its flanking sequences ( Fig. 1), we located the DNA region responsible for the lens-specific gene regulation within 136 base pairs (bp) encompassing the transcription initiation site (from nucleotide positions -81 to + 55) (9). Most remarkably, deletion of the segment from -81 to -65 reduced the level of expression 20-fold. This segment contains the GC box sequence ( Fig. 1) which is often part of viral and animal gene promoters as an essential cis-acting element for transcription initiation (5-7, 10, 15). To characterize regulatory interactions of putative trans-acting cellular factors with the cisacting DNA element of the 5-crystallin gene, we analyzed competition between the nuclear-injected gene and various coinjected DNA fragments.First, a 72-bp fragment of the promoter region (from positions -107 to -36) of the B-crystallin gene (fragment A, Fig. 2B) was tested for its effect on b-crystallin expression upon coinjection with the gene. Two test genes were used: one was the 5-crystallin gene on plasmid pSClB (12) and the other was a chimeric gene (Mov-8 gene) on p5Kp-Mov (11) in which the Moloney murine leukemia virus (MoMuLV) long terminal repeat (LTR) (22) was linked to the 5-crystallin gene at the second intron. The former directs the synthesis of a 50-kilodalton polypeptide (Fig. 3A, lane 1), and the latter directs the synthesis of 48 and 51-kilodalton polypeptides (11) (Fig. 3A, lane 3); therefore, their products could be separately analyzed on an immunoblot (Fig. 3A, lane 2). A mixture of both test genes (125 pLg of each per ml) and fragment A (500 ,ug/ml) was injected into the nuclei of mouse lens epithelial cells in primary culture (13). Assuming an average injection volume of 10 fl (1,3,8), we estimated the numbers of injected competitor fragments and each of the test genes to be 6 x 104 and 60 per nucleus, respectively. The injected cells were harvested 48 h later, and expression of the test genes was examined (Fig. 3A, lane 4) (Fig. 3B). It is difficult to estimate the actual number of regulatory-factor molecules which may bind to the fragment A region of the 5-crystallin promoter, but...

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confidence: 99%

In vivo competition of delta-crystallin gene expression by DNA fragments containing a GC box.

Hayashi¹,

Kondoh²

1986

Mol. Cell. Biol.

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“…Negligible CAT gene expression was present when pSVO-CAT (no promoter) (14) or paA88a-CAT was used for transfection (15). Neither paoA366a-CAT nor paA88a-CAT was functional in nonlens cells, indicating that Pr activation of CAT gene expression displays considerable lens specificity (15).…”

Section: Introductionmentioning

confidence: 99%

“…3) were transfected with paA366a-CAT, CAT gene expression was observed (15). Negligible CAT gene expression was present when pSVO-CAT (no promoter) (14) or paA88a-CAT was used for transfection (15).…”

Section: Introductionmentioning

confidence: 99%

“…The resulting plasmids were called poLA3662-CAT and paA882-CAT, respectively. The subscript "a" signifies that the plasmids contain the murine DNA fragment in the same orientation as in the original gene (15).…”

Section: Introductionmentioning

confidence: 99%

“…The plasmids were introduced into the explants by the calcium phosphate method (15) and CAT assays were performed 3 days later. This system has been very useful for studying crystallin gene regulatory signals (15,16).…”

Section: Introductionmentioning

confidence: 99%

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Crystallin genes: lens specificity of the murine alpha A-crystallin gene.

Chepelinsky

Khillan

Mahon

et al. 1987

Environ Health Perspect

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The abundant soluble proteins of the eye lens, the crystallins, are encoded by several gene families which are developmentally regulated in the embryonic lens. We have studied the expression of the murine aA-crystallin gene. Transfection experiments using the pSVO-CAT vector and explanted lens epithelia from embryonic chickens demonstrated proximal (-88 to -60) and distal (-111 to -85) regulatory sequences which interact when the aA-crystallin promoter is activated in the lens cells. Transgenic mouse experiments showed that the sequence between positions -366 to + 46 of the aA-crystallin promoter can drive foreign genes selectively in the lens. A fusion gene consisting of this aA-crystallin promoter sequence and the T-antigen gene of SV40 produced a lens tumor in transgenic mice. Thus, crystallin promoters provide a useful model for tissue-specific gene expression and permit targeting the expression of foreign genes to a highly differentiated tissue during development.

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Regression of vessels in the tunica vasculosa lentis is initiated by coordinated endothelial apoptosis: A role for vascular endothelial growth factor as a survival factor for endothelium

1998

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Lens-specific expression of the chloramphenicol acetyltransferase gene promoted by 5' flanking sequences of the murine alpha A-crystallin gene in explanted chicken lens epithelia.

Cited by 119 publications

References 42 publications

In vivo competition of delta-crystallin gene expression by DNA fragments containing a GC box.

In vivo competition of delta-crystallin gene expression by DNA fragments containing a GC box.

Crystallin genes: lens specificity of the murine alpha A-crystallin gene.

Regression of vessels in the tunica vasculosa lentis is initiated by coordinated endothelial apoptosis: A role for vascular endothelial growth factor as a survival factor for endothelium

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