Photolyase/cryptochrome family blue-light photoreceptors use light energy to repair DNA or set the circadian clock.
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Amino Acid Sequence↗
Biomedical subjects
R J Thresher
Publications and source records attributed to R J Thresher.
Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2.
Cryptochromes regulate the circadian clock in animals and plants. Humans and mice have two cryptochrome (Cry) genes. A previous study showed that mice lacking the Cry2 gene had reduced sensitivity to acute light induction of the circadian gene mPer1 in the suprachiasmatic nucleus (SCN) and had an intrinsic period 1 hr longer than normal. In this study, Cry1(-/-) and Cry1(-/-)Cry2(-/-) mice were generated and their circadian clocks were analyzed at behavioral and molecular levels. Behaviorally, the Cry1(-/-) mice had a circadian period 1 hr shorter than wild type and the Cry1(-/-)Cry2(-/-) mice were arrhythmic in constant darkness (DD). Biochemically, acute light induction of mPer1 mRNA in the SCN was blunted in Cry1(-/-) and abolished in Cry1(-/-)Cry2(-/-) mice. In contrast, the acute light induction of mPer2 in the SCN was intact in Cry1(-/-) and Cry1(-/-)Cry2(-/-) animals. Importantly, in double mutants, mPer1 expression was constitutively elevated and no rhythmicity was detected in either 12-hr light/12-hr dark or DD, whereas mPer2 expression appeared rhythmic in 12-hr light/12-hr dark, but nonrhythmic in DD with intermediate levels. These results demonstrate that Cry1 and Cry2 are required for the normal expression of circadian behavioral rhythms, as well as circadian rhythms of mPer1 and mPer2 in the SCN. The differential regulation of mPer1 and mPer2 by light in Cry double mutants reveals a surprising complexity in the role of cryptochromes in mammals.
Animals↗
Role of mouse cryptochrome blue-light photoreceptor in circadian photoresponses.
Cryptochromes are photoactive pigments in the eye that have been proposed to function as circadian photopigments. Mice lacking the cryptochrome 2 blue-light photoreceptor gene (mCry2) were tested for circadian clock-related functions. The mutant mice had a lower sensitivity to acute light induction of mPer1 in the suprachiasmatic nucleus (SCN) but exhibited normal circadian oscillations of mPer1 and mCry1 messenger RNA in the SCN. Behaviorally, the mutants had an intrinsic circadian period about 1 hour longer than normal and exhibited high-amplitude phase shifts in response to light pulses administered at circadian time 17. These data are consistent with the hypothesis that CRY2 protein modulates circadian responses in mice and suggest that cryptochromes have a role in circadian photoreception in mammals.
Animals↗
Electron microscopic visualization of RecT protein and its complexes with DNA.
Electron microscopy has been used to examine Escherichia coli RecT protein alone and in the complexes it forms with DNA substrates, with which it catalyzes strand exchange in vitro. Negative staining has revealed that the 33 kDa RecT protein monomers form open C-shaped and closed O-shaped particles. RecT protein monomers assemble into donut-shaped oligomers containing seven or eight protein monomers and rod-like structures. When bound to single-stranded DNA, RecT forms highly twisted nucleoprotein filaments that are 18 nm in diameter and have a helical pitch of 10 nm. When added to linear duplex DNA in the presence of active RecE protein (exonuclease VIII), filamentous nucleoprotein complexes are formed on the DNA ends and the DNA molecules are frequently cyclized through protein-protein interactions.
Bacterial Proteins↗
Location of the origin of replication for the 7.5-kb Chlamydia trachomatis plasmid.
The hypothetical origin of replication for the 7.5-kb plasmid common to Chlamydia trachomatis is believed to be in a region of the plasmid that contains four 22-bp tandem repeats preceded by an A-T-rich region. To test this hypothesis, replication of plasmid DNA in metabolically active reticulate bodies of the Lymphogranuloma venereum biovar of C. trachomatis was examined by electron microscopy. The results presented show that the origin of replication appears to be near the tandem repeats of pCHL2. In addition, replication of the 7.5-kb plasmid is unidirectional, and the copy number during replication is 7-10. The evidence presented suggests that C. trachomatis has a homologue to the Escherichia coli dnaA gene and that this homologue might be involved in replication of the C. trachomatis 7.5-kb plasmid.
Base Sequence↗
NaeI endonuclease binding to pBR322 DNA induces looping.
Previous work has demonstrated the existence of both resistant and cleavable NaeI sites. Cleavable sites introduced on exogenous DNA can act in trans to increase the catalysis of NaeI endonuclease cleavage at resistant sites without affecting the apparent binding affinity of the enzyme for the resistant site [Conrad, M., & Topal, M. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. This activation suggests allosteric regulation of NaeI cleavage by distant cis- and trans-acting sites in DNAs containing both resistant and cleavable sites. Plasmid pBR322 contains four NaeI sites, at least one of which is resistant to cleavage. Electron microscopy is used here to demonstrate that NaeI endonuclease simultaneously binds to multiple recognition sites in pBR322 DNA to form loops with NaeI protein bound at the loop's base. The maximum number of loops formed with a common base suggests four binding sites per enzyme molecule. Looping was inhibited by addition of enzyme-saturating amounts of double-stranded oligonucleotide containing an NaeI site, whereas another double-strand oligonucleotide without the NaeI site had no effect. The number of loops seen was not above background when double-stranded M13 DNA, which contains only a single NaeI recognition site, was used as substrate.
DNA Restriction Enzymes↗
Inhibition of human immunodeficiency virus type 1 morphogenesis in T cells by alpha interferon.
Some murine retroviruses exhibit altered release of virus when cells are treated with alpha interferon (IFN-alpha), resulting in the accumulation of intracellular virions in cytoplasmic vacuoles. In studies of the inhibitory effect of IFN-alpha (Wellferon) on acute human immunodeficiency virus type 1 infection of human T-cell lines, we found that in C3 cells, the 50% effective concentration was 9 U/ml and the 90% effective concentration was 310 U/ml. There was no apparent accumulation of intracellular particles detected by p24 antigen levels or by processing the cells for electron microscopy. Extracellular reverse transcriptase activity and p24 levels decreased in parallel with increasing IFN, whereas the intracellular viral proteins decreased only slightly. By electron microscopy, cells treated with higher concentrations of IFN (512 U/ml) disclosed very few particles budding into extracellular spaces; no intracellular particles could be seen, despite nearly normal levels of intracellular viral protein detected by the p24 antigen assay and correct processing detected by Western blot (immunoblot) analysis. Thus in human immunodeficiency virus-infected cells, the major block produced by IFN-alpha appeared to be late in the viral cycle at the morphogenesis stage of virion production. Chronically infected Jurkat cells treated with IFN appeared to be inhibited in growth rate, as virus production decreased proportionally with cell number.
Acquired Immunodeficiency Syndrome↗
Intercalators promote the binding of RecA protein to double-stranded DNA.
Ethidium bromide, acridine orange, 4'-(9-acridinylamino)methanesulfon-o-anisidide (o-AMSA), and m-AMSA induce the rapid binding of RecA protein to double-stranded (ds) DNA. The filaments formed appear to retain the drug and are 12.8 nm in diameter with an 8.0-nm pitch. Two classes of drugs have been distinguished: (i) those that bind to RecA protein and induce assembly at low relative concentrations (e.g., ethidium bromide) and (ii) those that do not appear to interact directly with RecA protein and must be present at relatively high drug concentrations to stimulate assembly (e.g., m-AMSA). Ethidium bromide, acridine orange, and quinacrine inhibit RecA protein binding to single-stranded DNA. Addition of ATP to the drug-induced filaments causes the protein to rapidly dissociate from dsDNA, and protein binding to dsDNA diminishes upon extended exposure to room light. We suggest that the structure of the drug-induced filaments may be more typical of the complex that initiates RecA protein assembly along DNA rather than the product of extensive polymerization as induced by adenosine 5'-[gamma-thio]triphosphate.
Coliphages↗
Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA.
We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional RecA protein to yield presynaptic filaments. Here, electron microscopy has been used to further explore the parameters of this assembly process. The optimal extent of presynaptic filament formation required at least one RecA protein monomer per three nucleotides, high concentrations of ATP (greater than 3 mM in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein assembly.
Adenosine Triphosphate↗
Gas-mediated vitreous compression: an experimental alternative to mechanized vitrectomy.
We have developed a simple technique in rabbit and baboon eyes that utilizes intravitreally injected perfluoropropane gas, which expands slowly to efficiently compress and displace nearly completely the vitreous body. There is cataract formation after extended contact of the gas bubble with the lens. However, it is rapidly reversible by reducing the duration of lens contact (gas-fluid exchange) and by using young animals. No long-term alterations in intraocular pressure or retinal function were observed, as determined by electroretinography, during the 4-month test period. Gross examination and scanning electron microscopy revealed that the vitreous cavity, shortly after full expansion of the gas bubble, was practically free of collagen. The vitreous body had been detached from most of the retina and compressed into a collagenous strand between the optic nerve head and lens in the rabbit eyes, and there was also a dense collagen accumulation on the inferior retinal surface and anterior vitreous base in the rabbits and the baboon. Large areas of the retina and medullary wings were stripped of overlying collagen. By the end of 4 months, the compressed vitreous body had expanded to become an irregular structure that remained separated from the retina in areas of previous detachment. Mechanized vitrectomy is a difficult procedure often needed in experimental work. We believe that the vitreous compression and gas-fluid exchange technique is a valid alterative to a mechanical approach. We also believe that we have a model that simulates the human situation of posterior vitreous detachment and vitreous syneresis.
Animals↗
Vitreous hemorrhage nontoxic to retina as a stimulator of glial and fibrous proliferation.
To test the belief that blood is toxic to the retina, we developed an experimental model of vitreous hemorrhage in rabbits by injecting various amounts of autologous, uncoagulated , whole blood into their vitreous cavities, with the vitreous humor either intact or previously compressed by an expanding perfluoropropane gas bubble. Blood in the eyes with compressed vitreous cleared in half the time required for the same volume injected into eyes with intact vitreous (75.6 days vs 39.2 days). Large volumes of intravitreal blood (0.25 to 0.50 ml) were never toxic to the retina by ganzfeld and bright-flash electroretinography throughout a four-month observation period. The results indicated that massive vitreous hemorrhage has a dense filtering effect that can extinguish the ganzfeld but not the bright-flash electroretinogram. Blood caused moderate hemoglobin staining of the retina, without significant iron staining (confirmed by X-ray microprobe analysis). Hemoglobin residues accumulated within cells of the inner retina, especially Müller's cells. Blood clot retraction after the injection of large volumes of fresh blood (1 ml) produced traction retinal detachment, hole formation, and subretinal accumulation of blood. This correlated with a complete and persistent extinction of the ganzfeld and bright-flash electroretinograms throughout the four-month observation period. A striking finding was that almost all eyes developed glial membranes on the peripheral retina. Fibrous membranes, causing local retinal contraction, were found over the medullary wings and optic disk in eyes with vitreous compression.
Animals↗