Continuous Improvement  Continuous improvement is the philosophy of continually seeking ways to improve processes based on a Japanese concept called kaizen. 1. Train employees in the methods of statistical process control (SPC) and other tools. 2. Make SPC methods a normal aspect of operations. 3. Build work teams and encourage employee involvement. 4. Utilize problem-solving tools within the work teams. 5. Develop a sense of operator ownership in the process. © 2007 Pearson Education
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P16065 Antisense Copies of Light-Regulated Genes in Rice Abstract T Mike1, N Joshi1, J Bird1, K Margavage1,2, Bryant Morocho1, X-W Deng2, W Terzaghi1,2 Natural antisense transcripts (NATs) are RNAs complementary to sense RNAs that are known to play roles in gene regulation. We studied 21 genes with NAT that are involved in light-regulated pathways in Nipponbare rice (Oryza sativa japonica). Of these genes, 17 were detected by RT-PCR in shoots and roots of Nipponbare seedlings. RTPCR of the Os12g17600 rbcS gene detected multiple small antisense fragments rather than one continuous RNA. Quantitative RT-PCR of Os12g17600 identified 5-fold more sense than NAT in shoots of seedlings grown in light, but 14-fold more sense than NAT in darkgrown seedlings. qRT-PCR of the Os03g51030 PHYA gene indicated that all light treatments decreased the ratio of sense to antisense with the exception of far-red light, which increased the ratio. Several genes exhibited reciprocal regulation of NAT and sense RNAs according to light treatment. Low molecular weight RNA blots of the Os03g07300/ Os03g07310 gene pair identified a small RNA (~40 nucleotides) that was only observed in light-treated roots. These small RNAs might be used to down-regulate the expression of genes turned on by light in roots. 1 Wilkes University, Wilkes-Barre, PA - 2YaleUniversity, New Haven, CT Os03g07300/ Os03g07310 (ribulose-3-P epimerase/ axi protein) Os02g05830 (rbcS2) Conclusions  17 of 21 light-regulated genes examined have NATs, 5 of which are regulated by light  Several genes show reciprocal regulation of mRNA and NAT  Some NATs were processed into small RNAs which may help regulate sense/ antisense RNA transcription  qRT-PCR detected:  ~14-fold more rbcS mRNA than NAT transcripts in shoots grown in continuous darkness  ~5-fold more mRNA than NAT in shoots grown in continuous light  Roots grown 4hr in white light do not increase expression of rbcS mRNA  phyA NAT expression greatly increased upon exposure to white light for 4 Hr or to1mmol. m2 red light. Introduction Discussion  Natural Antisense Transcripts (NATs) RNA molecules complementary to other “sense” RNAs  Present in a variety of organisms, NATs are involved in RNA editing, genomic imprinting, viral defense, etc.  Sense-antisense RNA pairs may reciprocally regulate each other’s production: when production of one transcript increases, production of the other decreases  Prevalence of NATs in plants suggests that NATs may help regulate light responses  Light responses are regulated by complex networks of transcripts  Some antisense RNA is involved in circadian rhythms  Although NATs have been identified in model plant species, their functions are not clear  Tiling-path microarrays identified thousands of genes with NATs   17 of 21 NATs of light-regulated genes found by microarrays were confirmed, validating this high-throughput approach.  Reciprocal light regulation of sense and antisense transcripts was detected for several genes, providing a potential mechanism for regulating the abundance of specific transcripts in response to light. Figure 2: The Os03g07300/ Os03g07310 (ribulose-3-P epimerase/ axi protein) gene pair. A) Low molecular weight Northern showing a 40 nt, root-specific RNA derived from Os03g07310. B) RT-PCR confirming the presence of mRNA of both genes in the leaf tissues. LL: light-grown leaf; DL: dark-grown leaf.  Overlapping NATs initiated from several start sites were identified for Figure 3: Reciprocal regulation of Os02g05830 (rbcS2). In tissues expressing higher levels of NAT, the mRNA is found at lower levels, and vice versa, indicating reciprocal regulation. Os12g17600  Suggests that antisense is not initiated from a single promoter.  Small RNAs derived from several overlapping gene pairs were detected, which may help regulate their expression. LL: light leaf; DL: dark leaf; LR: light root; DR: dark root; 4hr. WL: 4 hour white light; 4hr. WR: 4 hour white root; RL: red leaf; RR: red root; FRL: far red leaf; FRR: far red root; BL: blue leaf; BR: blue root.  NATs are induced in greater magnitudes than sense mRNA in both rbcS and phyA leaves under various light treatments  Questions that still need answers:   Os03g51030 (PHYA) Os12g17600 (rbcS)    Which photoreceptors are involved? Do NATs help modulate light-regulated gene expression? How is NAT/ mRNA production regulated? Are NATs polyadenylated? Sequence of 40nt RNA product of 07300 gene? Methods Figure 1: Antisense and light regulation. High-throughput techniques identified large numbers of antisense and lightregulated transcripts. This research tested the hypothesis that antisense may play a role in light regulation.  Identified antisense transcripts from light-regulated genes in Japonica rice  Query microarray, MPSS, and cDNA databases  Treated seedlings to a variety of lighting conditions to determine effect on mRNA and antisense transcription Plants were grown: 10 days continuous white light or continuous darkness  10 days continuous darkness followed by 4 hours white light  10 days continuous darkness followed by either 1 mmol red light, 1 mmol far red light, or 1 mmol blue, then far red  RNA was extracted from roots and leaves using Ambion’s miRvana Total RNA Isolation kit.  Detection of mRNA and antisense utilized:  Northern blots to verify presence of RNA  Reverse Transcription using the 5’ or 3’ primer only  Real time PCR to quantify relative expression   Expression of Sense and Antisense phyA Transcripts Relative to DLAS in Shoots B) Table 1: Strength of detected antisense signals. The gene pairs in the red box overlap at their annotated 3’ ends and the snoRNA are transcribed from the opposite strands of RPT2 exons. The antisense strands of the remaining genes have no annotated functions. Blue Dark Far Red Light Red 4 hr White Antisense Sense Ratio S:A 1.2 156.0 130.9 1.0 152.1 152.1 0.8 132.2 172.6 2.0 81.9 41.8 4.9 112.3 23.0 2.5 186.9 75.3 B) Expression of Sense and Antisense rbcS Transcripts Relative to DLAS in Shoots Blue Dark Far Red Light Red 4 hr White Antisense 5.8 1.0 Sense Ratio S:A 85.3 14.7 13.9 13.9 5.5 100.6 11.0 57.4 527.3 122.5 10.4 5.2 11.2 40.2 284.0 7.1 Expression of Sense and Antisense rbcS Transcripts Relative to DRAS in Roots Blue Dark Far Red Light Red 4 hr White Antisense 0.4 1.0 Sense Ratio S:A 5.5 12.5 9.8 9.8 1.2 2418.4 153.8 15.7 16842.5 1854.2 12.9 7.0 12.1 0.1 2.5 19.4 Figure 4: Induction of Os03g51030 (phyA) in seedling shoots by various light treatments. Figure 5: Induction of Os12g17600 (rbcS) in seedling shoots by various light treatments. A) Graph of level of induction of sense and antisense standardized to the corresponding dark sample. A)Graph of level of induction of sense and antisense standardized to the corresponding dark sample. B) Expression levels of sense and antisense RNA in shoots relative to dark shoot antisense along with the ratio of sense to antisense. B)Expression levels of sense and antisense RNA in shoots (left) relative to dark shoot antisense and roots (right) relative to dark root antisense along with the ratio of sense to antisense. Acknowledgements This research was primarily supported by NSF grant DBI-0421675: Virtual Center for Analysis of Rice Genome Transcription (XingWang Deng, PI). Additional support from Wilkes University, Yale University, and the Howard Hughes Medical Institute is also gratefully acknowledged.
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Lactation Support Program ELIGIBILITY & REQUIREMENTS Eligible Employees & Students Any Pasadena Area Community College District employee or student desiring to express breast milk shall be accommodated, and will be eligible for use of designated Lactation Support Program (LSP) rooms. Human Resources Home (link) Location The District shall make reasonable efforts to provide nursing employees and students with the use of an LSP room or other location, other than a toilet stall or locker room, to express in private. The room or location shall be shielded from view and free from intrusion of others. The rooms will be centrally located to accommodate employees and students that work or attend classes in various buildings; however, the rooms will not necessarily be located in the building in which the employee works or student attends class. An employee assigned to a private unshared office may use that room. Designated LSP rooms shall contain a supportive chair, a table, access to an electrical outlet, adequate ventilation, and a door that can be locked. Room Access Access to LSP room will be unscheduled and on a first–come, first-served basis. Any employee who wishes to use an LSP room must first contact Human Resources to arrange lactation support accommodations, and to be provided instructions for use of and access to the room. Students must contact the Student Health Services office to receive instructions regarding use of the student LSP room. Use of Room The LSP rooms are for the exclusive use of nursing employees and students, on a first–come, first-served basis, and one at a time. Each nursing employee or student user of the room is responsible for keeping the room clean and removing personal items when leaving. If an employee or student finds the room in disarray, she is to contact the Human Resources Office or Student Health Services Office. Employee Break Time The District shall provide a reasonable amount of unpaid break time to accommodate an employee who desires to express breast milk. The supervisor shall permit the employee to take breaks to express whenever and as often as needed. Whenever possible, the break time shall run concurrent with any break time already provided to the employee. For example, employees who work 5 hours or more are provided two (2) twenty (20) minute paid rest breaks and one (1) one-half (1/2) hour unpaid meal break; employees who work less than 5 hours are provided one (1) twenty 20 minute paid rest break. Therefore, an employee who works eight (8) hours would have three (3) provided breaks; and an employee who works four (4) hours would have one (1) provided break. An employee may need other breaks, in addition to the provided breaks. The frequency of the breaks needed by an employee, as well as the duration, may vary. Termination of Accommodation An employee or student may use the LSP room for as long as needed. When an employee no longer has need of lactation support, she must inform Human Resources and her supervisor, and return to her regular break schedule. When a student no longer has need of lactation support, she must inform the Student Health Services Office. LSP Introduction (link) Eligibility & Requirements (link) FLSA Nursing Mothers Rights (link) LSP Accommodation Request Form (link) Designated Locations (link) Campus Map (link)
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