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This page talks about the Per gene in Drosophila and the associated Per1, Per2, and Per3 genes in mammals. The functions of the aforementioned period genes in both circadian and non-circadian contexts are explored as well as their clinical significance.

Drosophila Per Gene
Period (Per) is a gene located on the X chromosome of Drosophila melanogaster. Oscillations in levels of both Per transcript and its corresponding protein PER have a period of approximately 24 hours and together play a central role in the molecular mechanism of the Drosophila biological clock (Drosophila circadian rhythm) driving circadian rhythms in eclosion and locomotor activity. Mutations in the Per gene can shorten (PerS), lengthen (PerL), and even abolish (Per01) the period of the circadian rhythm.

Discovery of Drosophila Per
In 1971, Ronald J. Konopka and Seymour Benzer discovered Drosophila Per and Drosophila Per mutants. Konopka and Benzer isolated the Period gene and three associated mutants (PerS, PerL, and Per01) using an EMS mutagenesis screen. The PerS, PerL, and Per01 mutations were found to not complement each other, so it was concluded that the three phenotypes were due to mutations in the same gene. The PerS allele has an intrinsic period of around 19 hours. The PerL allele has an intrinsic period of around 29 hours, and the Per01 allele is aperiodic. In 1984 scientist Michael Rosbash cloned the Per gene, and by 1988 proved that the PER protein is circadian in nature, as it is rhythmic in the light dark cycle and constant darkness. The discovery of mutants that altered the period of circadian rhythms in eclosion and locomotor activity (PerS and PerL) indicated the role of the Per gene in the clock itself and not an output pathway. Experimentation in 1990 by Rosbash, Jeffrey Hall and Paul Hardin revealed that the Per01 mutant allele produces a non-functional protein transcript. Meanwhile, when PerL and PerS are inserted into the null (Per01) mutant host, rhythmicity of the donor organism is restored in the mutant host. The experimental results also showed that when Per01 flies were transformed using a 7.2kb piece of functional Per DNA, the inserted Per7.2 encodes for a functional Per mRNA as well as a function PER protein. The discovery of oscillating levels of period mRNA and associated PER proteins in the research by Rosbash, Hall, and Hardin generated evidence of a negative feedback loop in Drosophila that controlled circadian rhythmicity, as PER protein represses the activity of the period gene. In 1990 Rosbash, Hall and Hardin officially proposed the transcriptional translational feedback loop as the mechanism for the circadian clock. In 1998, it was discovered that Per produces two transcripts (differing only by the alternative splicing of a single untranslated intron) which both encode the PER protein. These genes were differentiated by Albrecht and Sun by subjecting the transcripts to various different light dark conditions. They discovered that both Per1 and Per2 maintain expression in constant darkness, but can entrain to new light dark cycles. The differentiating factor became the induction of the Per2 gene in the Suprachiasmatic nucleus (SCN) at CT 22. CT, or circadian time, is a standardized 24 hour period of an organism’s circadian cycle without reference to external cues.

Drosophila Per Circadian Function
In Drosophila, Per mRNA levels oscillate with a period of approximately 24 hours, peaking at CT 16 in a 12:12 LD cycle or 4 hours after lights off in photoperiods less than 16 hours. PER protein also oscillates with a nearly 24-hour period, peaking about six hours after peak Per mRNA levels. When PER levels increase, Per transcription inhibition increases, lowering protein levels. However, because PER protein cannot directly bind to DNA, PER protein does not directly influence its own transcription; alternatively, it inhibits its own activators. After PER is produced from Per mRNA, it dimerizes with Timeless (TIM) and the complex goes into the nucleus and inhibits the transcription factors of per and tim, the CLOCK/CYCLE heterodimer. This CLOCK/CYCLE complex acts as a transcriptional activator for Per and Tim by binding to specific enhancers (called E-boxes) of their promoters. Therefore, inhibition of CLK/CYC lowers Per and Tim mRNA levels, which in turn lower the levels of PER and TIM. Now, cryptochrome (CRY) is a light sensitive protein which inhibits TIM in the presence of light. When TIM is not complexed with PER, another protein, doubletime (DBT)phosphorylates PER, targeting it for degradation.

Drosophila Per Non-Circadian Function
In Drosophilia melanogaster, Per has been shown to have a couple non-circadian clock functions.

First, Per has been shown to be necessary and sufficient for long-term memory (LTM) formation in Drosophila melanogaster. Per mutants show deficiencies in LTM formation that can be rescued with the insertion of a Per transgene and enhanced with overexpression of the Per gene. This response is absent in mutations of other clock genes (timeless, dClock, and cycle). Research suggests that synaptic transmission through Per-expressing cells is necessary for LTM retrieval.

Second, Per has been shown to extend the lifespan of the fruit fly, suggesting a role in aging. This result, however, is still controversial, as the experiments have not been successfully repeated by another research group.

Third, Per has been shown to be necessary and sufficient to govern female-mating Drosophila melanogaster activity. Drosophila melanogaster displays circadian rhythm in their mating, leading to mating at a specific time in the day. However, Per1 mutations resulted in a loss of that circadian rhythm, thus allowing researchers to conclude that clock genes govern the circadian rhythm of female mating activity.

Fourth, Per has been shown to be necessary and sufficient for maintaining the physiological well-being of Drosophila melanogaster by regulating response to oxidative stress. Per knockout experiment showed increase susceptibility to H2O2 compared to wild-type flies. This result coincided with increased mitochondrial H2O2 in the Per knockout mice which resulted in oxidative damage. These results show that Per is essential for anti-oxidative defense.

Mammalian Per Gene
Per1, Per2 and Per3 are part of the mammalian period gene family, expressed in the suprachiasmatic nucleus (SCN) of the mammalian brain. The SCN acts as the primary circadian pacemaker in mammals. Per1 and Per2 genes are clock genes, necessary for upkeeping circadian rhythmicity and light responsiveness in the SCN. Similar to Per1 and Per2, Per3 oscillates with a circadian rhythm in the SCN and other peripheral tissues, including skeletal muscles and the liver. Unlike Per1 and Per2, Per3 has no responsiveness to light, therefore its role in the transcription- translation feedback loop has not been concretely established.

Discovery of Mammalian Per
Mammalian orthologs of the Drosophila Per gene, Per1, Per2 and Per3 were discovered and cloned by scientists Hajime Tei and Hitoshi Okamura in 1997-1998. Through experimentation, the function of Per1 and Per2 as clock genes was discovered, as Per1 increase led to a decrease in intrinsic period, Per2 increase led to an increase in intrinsic period, and a knockout of both Per1 and Per2 yielded arrhythmicity in the host mammal. Per3 gene was found not to be a clock gene as it had no phenotype.

Mammalian Per Circadian Function
In mammals, an analogous transcription-translation negative feedback loop (TTFL) to the Drosophilia is observed. Translated from the three mammalian homologs of Drosophila-per, one of three PER proteins (PER1, PER2, and PER3) dimerizes via its PAS domain with one of two cryptochrome proteins (CRY1 and CRY2) to form a negative element of the clock. This PER/CRY complex moves into the nucleus upon phosphorylation by CK1-epsilon (casein kinase 1 epsilon) and inhibits the CLK/BMAL1 heterodimer, the transcription factor that is bound to the E-boxes of the three per and two cry promoters by basic helix-loop-helix (BHLH) DNA-binding domains.

The mammalian Per1 and Per2 genes play key roles in photoentrainment of the circadian clock to light pulses. This was first seen in 1999, when Akiyama et al. showed that mPer1 is necessary for phase shifts induced by light or glutamate release. Two years later, Albrecht et al. found genetic evidence to support this result when they discovered that mPer1 mutants are not able to advance the clock in response to a late-night light pulse (ZT22) and that mPer2 mutants are not able to delay the clock in response to an early night light pulse (ZT14). Thus, mPer1 and mPer2 are necessary for the daily resetting of the circadian clock to normal environmental light cues.

Mammalian Per Non-Circadian Function
The mammalian Per2 gene plays a key role in tumor suppression. Mice with an mPer2 knockout show a significant increase in tumor development and a significant decrease in apoptosis. This is thought to be caused by mPer2 circadian deregulation of common tumor suppression and cell cycle regulation genes, such as Cyclin D1, Cyclin A, Mdm-2, and Gadd45α, as well as the transcription factor c-myc, which is directly controlled by circadian regulators through E box-mediated reactions.

In addition, mPer2 knockout mice show increased sensitivity to gamma radiation and tumor development, further implicating mPer2 in cancer development through its regulation of DNA damage-responsive pathways. Thus, circadian control of clock controlled genes that function in cell growth control and DNA damage response may affect the development of cancer in vivo.

Homologs of Per
Homologs can either be orthologs or paralogs. Orthologs are homologous genes that usually have similar function in two different species, arising from a common ancestor. Paralogs are homologous genes that arose through gene duplication within a species, they usually have slightly different functions. On a similar note, circadian clocks also display robustness, or insensitivity to changes in the system. Per is thought to be present in the last common ancestor of all animals. The African clawed frog, the Tropical clawed frog, Lizards, Rainbow trout, Worms, and Zebrafishes among other animals have at least one ortholog to either mammalian Per1, Per2, or Per3. However, various plant groups such as tomatoes, wheat, barley, and sugarcane, do not express an ortholog to Per. The mammalian molecular clock orthologs to drosophila Per are: Per 1, Per 2, and Per 3. Per 1 and Per 2 are paralogs. This redundancy between M. Per1 and M. Per2 reinforces the robustness of the system, because if either one was knocked out, the other would compensate by increasing concentration. undefinedThe human Per orthologs show sequence similarity to Drosophila Per and also contain the PAS domain and nuclear localization sequences that the Drosophila Per has. The human Per proteins are expressed rhythmically in the suprachiasmatic nucleus as well as areas outside the SCN. Additionally, while Drosophila PER (Per protein) moves between the cytoplasm and the nucleus, mammalian PER is more compartmentalized: mPer1 primarily localizes to the nucleus and mPer2 to the cytoplasm.

Familial Advanced Sleep-Phase Syndrome
Familial advanced sleep-phase syndrome (FASPS) is known to be associated with mutations in the mammalian Per2 gene. FASPS is most commonly attributed to a single nucleotide exchange that replaces Serine 662 with Glycine in the Ck1e phosphorylation binding site on Per2. This point mutation causes hypo-phosphorylation of PER2 protein, reducing its stability. FASPS is characterized by a short endogenous period and about a 4 hour advanced phase. People with this syndrome usually go to sleep in the early evening (around 7pm) and wake up before sunrise (around 4am). Chronotherapy is used as a treatment in an attempt to alter the phase of the individual's clock using cycles of bright light.

Delayed Sleep Phase Syndrome
Delayed sleep phase syndrome is characterized by regular, but delayed sleep-onset time, and delayed dim light melatonin onset (DLMO). Onset occurs most frequently in adolescence with about 7% of the adolescent population suffering from this syndrome. Though the syndrome has a large environmental component, it is also associated with polymorphisms of Per3 gene; 40% of syndrome sufferers have a family history of the syndrome.

Tumorigenesis
Past research has found significantly downregulated Per1 and 2 mRNA expression levels in prostate, breast, gastric, pancreatic, and colorectal cancer, melanoma, chronic lymphocytic leukemia, and hepatocellular carcinoma. Per3 expression is also greatly reduced in pancreatic and colorectal cancer, and hepatocellular carcinoma. Extent of decrease in expression of Per1, 2, and 3 has been shown to be negatively correlated with survival time, and positively correlated with chances of metastasis.

Cirrhosis
Cirrhosis is a late stage liver scarring disease associated with liver portal vein hypertension, a delayed circadian cycle, and changes in melatonin levels. The effects of Per mRNA expression on this disease has been studied in mice. Global Per2 knockout mice develop a more severe form of cirrhosis than control populations, and Per1, Per2 double-knockout mice show increased hepatic bile acid levels, which leads to cholestasis, a causing factor for cirrhosis.

Adrenal Disorders
Many clock genes and proteins have been studied in relation to endocrine disorders, particularly in mice and rats. The human adrenal gland expresses Per1, Per2, Cry2, Clock, and Bmal1. Adrenocorticotropic Hormone (ACTH), the hormone released from the pituitary gland that promotes cortisol release from the adrenal gland, increases Per1 mRNA levels; this positive regulation is inhibited by the addition of melatonin. Most research on circadian clock effects on adrenal disorders has been limited to animal studies. 30 minute light pulses were found to increase Per1 mRNA and Per2 mRNA in all renal cortical layers. Further, a light-induced increase in corticosterone was observed, independent of any change in ACTH. Further notice should be given to the fact that the SCN and the pituitary gland are located very close to each other and this spatial proximity is thought to aid in coupling the two systems.

Though many clinical studies on Clock genes are limited to murine and rat models, they provide evidence for how pervasive and pertinent clock genes, such as Per, are in maintaining a healthy body state. In addition, these studies offer new therapeutic targets for further research.