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.     

In vivo persistence of DNA triple helices containing psoralen-conjugated oligodeoxyribonucleotides.

Triple helices represent an attractive method for modulating specific gene expression. In particular, cross-linking between a triplex-forming oligonucleotide (TFO) and its duplex DNA target, typically through the formation of psoralen photoadducts, allows efficient blocking of elongation by RNA polymerases in vitro. However, in vivo, this approach is limited by DNA repair of the photoadduct. Here we describe the use of an oligodeoxyribonucleotide 19mer psoralen-modified TFO to form covalent linkages between an oligonucleotide and both strands of the targeted duplex DNA, thereby efficiently blocking expression of a luciferase reporter gene. Most importantly, we demonstrate that both the psoralen cross-link and the purine-motif triplex remained intact for at least 72 h post-transfection, indicating that such species can persist for an extended period of time in vivo. These findings support the feasibility of an antigene approach for the therapeutic regulation of specific gene expression.     

In vivo inhibition of TonB-dependent processes by a TonB box consensus pentapeptide.

The TonB box, a conserved pentapeptide sequence found in TonB-dependent colicins and receptors, is thought to interact physically with the TonB protein to facilitate TonB-dependent processes. Strains of Escherichia coli were treated in vivo with the synthetic TonB box pentapeptide Glu-Thr-Val-Ile-Val. The pentapeptide inhibited several TonB-dependent processes, including cell growth in low-iron medium, phi 80 infection, and killing by colicins B and Ia. Two unrelated control pentapeptides had no effect on TonB-dependent processes.     

Recognition and structural perturbation of GC box DNA by Sp1 zinc finger.

Interaction of Sp1 with GC box DNA was investigated by several footprinting experiments. Methylation of four guanine bases is strongly protected by Sp1 binding, while one guanine base in GC box is extremely hypermethylated. Sp1 binding also induces new cleavage at 5'-GA-3' site within GC box by bleomycin-iron complex.     

In vivo gene delivery and expression by bacteriophage lambda vectors

AIMS: Bacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo. METHODS AND RESULTS: Mice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo. Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer. CONCLUSIONS: Unmodified lambda phage particles are capable of transducing mammalian cells in vivo, and may be taken up -- at least in part -- by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo, and suggest new approaches that may enhance the efficiency of this process.     

Chicken lens delta-crystallin gene expression and methylation in several non-lens tissues.

RNA sequences coding for the most abundant chicken lens proteins, delta-crystallin, were detected at very low levels in day old post hatch chick lung, heart, kidney and liver, and in 6 day embryo headless bodies. The pattern of cytosine methylation within the CCGG sequences of the delta-crystallin genes was also examined and shown to vary in several non-lens tissues, from several stages of development. Embryonic neural retina, which expresses a higher level of delta-crystallin RNA than the above tissues, is no less methylated in the sites studied than the tissues which have no association with the eye, and is actually more heavily methylated than the kidney. Thus no obvious correlation was found between undermethylation and gene expression.     

In vivo resolution of circular plasmids containing concatemer junction fragments from minute virus of mice DNA and their subsequent replication as linear molecules.

During replication of their linear, single-stranded DNA genomes, parvoviruses generate a series of concatemeric duplex intermediates. We have cloned, into Escherichia coli plasmids, junction fragments from these palindromic concatemers of minute virus of mice DNA spanning both the right end-to-right end (viral 5' to 5') and left end-to-left end (viral 3' to 3') fusions. When mouse cells were transfected with these circular plasmids and superinfected with minute virus of mice, the viral junctions were resolved and the plasmids replicated as linear chromosomes with vector DNA in their centers and viral DNA at their termini. Resolution did not occur when the concatemer joint was replaced by a different palindromic sequence or when the transfected cells were not superinfected, indicating the presence of latent origins of replication which could only be activated by a viral trans-acting factor(s). Moreover, the products of resolution and replication from the two termini were characteristically different. Analysis of individual terminal fragments showed that viral 5' (right-end) sequences were resolved predominantly into "extended" structures with covalently associated copies of the virally encoded NS-1 polypeptide, while bridges derived from the 3' (left) end resolved into both NS-1-associated extended termini and lower-molecular-weight "turn-around" forms in which the two DNA strands were covalently continuous. This pattern of resolution exactly coincides with that seen at the two termini of replicative-form intermediates in normal virus infections. These results demonstrate that the bridge structures are authentic substrates for resolution and indicate that the frequency with which extended versus turn-around forms of each terminus are generated is an intrinsic property of the telomere.     

In vivo and ex vivo regulation of bacterial virulence gene expression

Bacteria are remarkably adaptable organisms that are able to survive and multiply in diverse and sometimes hostile environments. Adaptability is determined by the complement of genetic information available to an organism and by the mechanisms that control gene expression. In general, gene products conferring a growth or survival advantage in a particular situation are expressed, while unnecessary or deleterious functions are not. Expression of virulence gene products that allow pathogenic bacteria to multiply on and within host cells and tissues are no exception to this rule. Being of little or no use to the bacterium except during specific stages of the infectious cycle, these accessory factors are nearly always subject to tight and coordinate regulation. As a result of recent advances, we are beginning to appreciate the complexities of the interactions between bacteria and their hosts. The ability to probe virulence gene regulation in vivo has broadened our perspectives on pathogenesis.     

In vitro packaging of a lambda Dam vector containing EcoRI DNA fragments of Escherichia coli and phage P1.

In this report we describe a coliphage lambda vector system for cloning endo R. EcoRI DNA fragments. This system differs significantly from those previously described in two ways. First, restricted and ligated DNA is encapsidated in vitro. Second, with increasing lambda DNA size in the range 78 to 100% that of wild-type, the efficiency of DNA encapsidation into infectious phage particles markedly increases. For lambda wild-type DNA the efficiency of in vitro packaging (10(6) to 10(7) plaques produced per microgram of added DNA) is equal to, or better than, the standard CaCl2 transfection method. The use of a Dam mutation to facilitate recognition of size classes of inserted fragments is described. Using this vector and in vitro packaging, several E. coli and phage P1 and R.EcoRI fragments were cloned.     

In vivo expression of a nonselected gene transferred into murine hematopoietic stem cells by electroporation

Mouse bone marrow cells were subjected to electroporation in the presence of RSVCAT and SV2NEO plasmids. CAT activity was detected in the G-418 resistant granulocyte-macrophage colonies. RSVCAT electroporated into primary bone marrow cells, repopulated lethally irradiated mice as demonstrated by the persistence of CAT activity in the hematopoietic tissues showing that electroporation can offer a powerful mode of gene transfer into bone marrow cells.     

Characterization of trans-acting factor(s) regulating beta-globin gene expression by in vivo competition

A beta-globin/TK fusion gene was microinjected into non-erythroid cells (Ltk- cells) and erythroid cells (murine erythroleukemia (MEL) cells), and the interactions of the regulatory cellular factors with the beta-globin sequences were investigated by the in vivo competition experiment. The fusion gene was expressed efficiently in Ltk- cells. This expression was inhibited by a co-injection with a three-fold molar excess of the 5'-flanking sequence of the beta-globin gene or with a nine-fold molar excess of the mammary tumor virus LTR, but not with the alpha-globin gene. The fusion gene was expressed very poorly in the uninduced MEL cells and highly in the induced MEL cells. The co-injection of the beta-globin gene did not affect expression in the MEL cells in either uninduced or induced conditions.     

In vivo and in vitro inhibition of JE gene expression by glucocorticoids

Glucocorticoids are potent anti-inflammatory agents which affect cell growth and migration in a wide variety of systems and have profound effects on monocytes, decreasing their circulating number as well as inhibiting their accumulation at sites of inflammation and injury. Although the mechanisms by which glucocorticoids regulate gene induction have been established, the mechanisms by which they inhibit inflammation or cell growth and migration have yet to been determined. JE is one of the most abundant genes induced by platelet-derived growth factor (PDGF) in vitro and is also induced in vivo in response to ischemia or injury. JE encodes a low molecular weight glycoprotein that functions in part as a monocyte chemotactic factor and thus may be important in recruiting monocytes to sites of tissue injury and/or inflammation. We report that glucocorticoids block the induction of JE mRNA by serum or PDGF in cultured vascular smooth muscle cells. The effect of glucocorticoids appears largely due to destabilization of JE mRNA and has specificity for JE, in that other "early" PDGF-inducible genes are not inhibited by glucocorticoids. The effect of glucocorticoids also occurs in vivo: methyl prednisolone blocks the constitutive expression and inhibits the ischemia-induced elevation of JE mRNA levels in rat kidneys. The inhibition of JE mRNA accumulation by glucocorticoids may be related to the anti-inflammatory effects of these agents and defines JE as a member of what may be a group of PDGF-inducible genes that are responsive to corticosteroids.     

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.     

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.     

Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine.

Using CD spectroscopy, guanine tetraplex formation was studied with short DNA fragments in which cytosine residues were systematically added to runs of guanine either at the 5' or 3' ends. Potassium cations induced the G-tetraplex more easily with fragments having the guanine run at the 5' end, which is just an opposite tendency to what was reported for (G+T) oligonucleotides. However, the present (G+C) fragments simultaneously adopted other conformers that complicated the analysis. We demonstrate that repeated freezing/thawing, performed at low ionic strength, is a suitable method to exclusively stabilize the tetraplex in the (G+C) DNA fragments. In contrast to KCl, the repeated freeze/thaw cycles better stabilized the tetraplex with fragments having the guanine run on the 3' end. The tendency of guanine blocks to generate the tetraplex destabilized the d(G5).d(C5) duplex whose strands dissociated, giving rise to a stable tetraplex of (dG5) and single-stranded (dC5). In contrast to d(G3C3) and d(G5C5), repeated freezing/thawing induced the tetraplex even with the self-complementary d(C3G3) or d(C5G5); hence the latter oligonucleotides preferred the tetraplex to the apparently very stable duplex. The tetraplexes only included guanine blocks while the 5' end cytosines interfered neither with the tetraplex formation nor the tetraplex structure.