In the transcription of prokaryotes, binding of RNA polymerase to the specific promoter region is achieved by:
Initiation of transcription in prokaryotes begins with the recognition of DNA sequences by RNA polymerase. First, the bacterial RNA polymerase catalyzes RNA synthesis through loose binding to any region in the double-stranded DNA and then through specific binding to the promoter region with the assistance of accessory proteins called a factors (sigma factors). A promoter region is the DNA region upstream of the transcription initiation site. RNA polymerase binds tightly at the promoter sites and causes the double-stranded DNA structure to unwind. Consequently, few nucleotides can be base-paired with the DNA template to begin transcription. Once transcription begins, the o factor is released. The growing RNA chain may begin to peel off as the chain elongates. This occurs in such a way that there are always about 10 to 12 nucleotides of the growing RNA chains that are base-paired with the DNA template.
When performing cell culture, cells should be:
Cell culture has become one of the most powerful tools in biomedical laboratories, as cultured cells are being used in a diversity of biologic fields ranging from biochemistry to molecular and cellular biology. Through their ability to be maintained in vitro, cells can be manipulated by the introduction of genes of interest (cell transfection) and be transferred into in vivo biologic receivers (cell transplantation) to study the biologic effect of the interested genes (Fig. below). In the common laboratory settings, cells are cultured either as a monolayer (in which cells grow as one layer on culture dishes) or in suspension. It is important to know the wealth of information concerning cell culturing before attempting the procedure. For example, conditions of culture will depend on the cell types to be cultured ( eg, origins of the cells such as epithelial or fibroblasts, or primary versus immortalized/transformed cells). It is also necessary to use cell type-specific culture medium that varies in combination of growth factors and serum concentrations. If primary cells are derived from human patients or animals, some commercial resources have a variety of culture media available for testing. Generally, cells are manipulated in a sterile hood, and the working surfaces are wiped with 70 to 80% ethyl alcohol solution. Cultured cells are usually maintained in a humidified carbon dioxide incubator at 37°C (98.6°F) and need to be examined daily under an inverted microscope to check for possible contamination and confluency (the area cells occupy on the dish). As a general rule, cells should be fed with fresh medium every 2 to 3 days and split when they reach confluency. Depending on the growth rate of cells, the actual time and number of plates required to split cells in two varies from cell line to cell line. Splitting a monolayer requires the detachment of cells from plates by using a trypsin or collagenase treatment, of which concentration and time period vary depending on cell lines. If cultured cells grow continuously in suspension, they are split or subcultured by dilution. Because cell lines may change their properties when cultured, it is not possible to maintain cell lines in culture indefinitely.
Cell culture and tra nsfection. A. Primary cells can be isolated from tissues and cultured in medium for a limited period of time. After genetic manipulations to overcome the cell aging process, primary cells can be immortalized into cell lines for long-term culture. B. DNA can be introduced into cells to produce recombinant gene products or to analyze the biologic functions of the gene.
All of the following are involved in gene regulation EXCEPT:
Living cells have the necessary machinery to enzymatically transcribe DNA into RNA and translate the mRNA into protein. This machinery accomplishes the two major steps required for gene expression in all organisms: transcription and translation (see Fig. below). However, gene regulation is far more complex, particularly in eukaryotic organisms. For example, many gene transcripts must be spliced to remove the intervening sequences. The sequences that are spliced off are called introns, which appear to be useless, but in fact may carry some regulatory information. The sequences that are joined together, and are eventually translated into protein, are called exons. Additional regulation of gene expression includes modification of mRNA, control of mRNA stability, and its nuclear export into cytoplasm (where it is assembled into ribosomes for translation). After mRNA is translated into protein, the levels and functions of the proteins can be further regulated posttranslationally.
Four major steps in the control of euka ryotic gene expression. Transcriptional and posttranscriptional control determine the level of messenger RNA (mRNA) that is available to make a protein, while translational and posttranslational control determine the final outcome of functional proteins. Note that posttranscriptional and posttranslational controls consist of several steps.
Which of the following is a regulator of the cell cycle?
The machinery that drives cell cycle progression is made up of a group of enzymes called CDKs. Cyclin expression fluctuates during the cell cycle, and cyclins are essential for CDK activities and form complexes with CDK. The cyclin A/CDKl and cyclin B/CDKl drive the progression for the M phase, while cyclin A/CDK2 is the primary S phase complex. Early G1 cyclin D/CDK4/6 or late Gl cyclin E/CDK2 controls the G1-S transition. There also are negative regulators for CDK termed CDK inhibitors, which inhibit the assembly or activity of the cyclin-CDK complex. Expression of cyclins and CDK inhibitors often is regulated by developmental and environmental factors.
Which of the following drugs is a monoclonal antibody to an oncogene?
Patients whose tumors overexpress HER-2/neu are candidates for anti-HER-2/neu therapy. Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody directed against HER-2/neu. Randomized clinical trials have demonstrated that single-agent trastuzumab therapy is an active and well-tolerated option for first-line treatment of women with HER-2/neu-overexpressing metastatic breast cancer. More recently, adjuvant trials demonstrated that trastuzumab also was highly effective in the treatment of women with earlystage breast cancer when used in combination with chemotherapy. Patients who received trastuzumab in combination with chemotherapy had between a 40 and 50% reduction in the risk of breast cancer recurrence and approximately a third reduction in breast cancer mortality.
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