DNA, organization, Replication, and repair. (Harpers)DNA in a eukaryotic cells is associated with a variety of proteins, resulting in a structure called chromatin.Much of the DNA is associated with histone proteins to form a structure called the nucleosome, Nucleosomes are composed of an octamer of histones around which about 150 bp of DNA is wrapped.Histones are subject to an extensive array of dynamic covalent modification that have important regulatory consequences.Nucleosomes and higher-order structures formed from them serve to compact the DNA.

DNA is transcriptionally active regions is sensitive to nuclease attack; some regions are exceptionally sensitive and are often found to contain transcription control sites.Highly transcriptionally active DNA (genes) is often clustered in regions of each chromosomes. Within these regions, genes may be separated by inactive DNA in nucleosomal structures. In eukaryotes the transcription unit-that portion of a gene that is copied by RNA polymerase-often consists of coding regions of DNA (exons) interrupted by intervening sequences of noncoding DNA (introns).

After transcription, during RNA processing, introns are removed and the exons are ligated together to form the mature RNA that appears in the cytoplasm; this process is termed RNA splicing.DNA in each chromosome is exactly replicated according to the rules of base pairing during the S phase of the cell cycle.

Each strand of the double helix is replicated simultaneously but by somewhat different mechanisms. A complex of proteins, including DNA polymerase, replicates the leading strand continuously in the 5 to-3 direction. The lagging strand is replicated discontinuously, in short pieces of 150-250 nucleotides, in the 3 to-5 direction

DNA replication occurs at several sites-called replication bubbles-in each chromosomes. The entire process takes about 9 h a typical cell and only occurs during the S phase of the cell cycle.A variety of mechanisms employing different enzymes repair damaged DNA, as after exposure to-chemical mutagens or ultraviolet radiation.

Synthesis of DNA. (Mark)Replication of the genome requires DNA synthesis.During replication, each of the two parental strands of DNA serves as a template for the synthesis of complementary strand.The site at which replication is occuring is called the replication fork.Helicases and topoisomerases are required to unwind the DNA helix of the parental strands.

DNA polymerase is the major enzyme involved in replication.DNA polymerase copies each parental template strand in the 3-to-5 direction, producing new strands in a 5-to-3 direction.The precursors for replication deoxyribonucleotide triphosphates.As DNA synthesis proceeds in the 5-to-3 direction, one parental strand is synthesized continuously, whereas the other exhibits discontinuous synthesis, creating small fragments that are subsequently joined, because of the requirement that DNA polymerase must synthesize DNA in the 5-to3 direction.

DNA polymerase requires a free 3-hydroxyl group of a nucleotide primer in order to replicate DNA. The primer is synthesized by the enzyme primase, which provides an RNA primer.The enzyme telomerase synthesizes the replication of the ends of linear chromosomes (telomeres).Errors during replication can lead to mutations, so error checking and repair systems function to maintain the integrity of the genome.

RNA synthesis, processing & modification.(Harpers)RNA is synthesized from a DNA template by the enzyme RNA polymerase.There are three distinct nuclear DNA-dependent RNA polymerases; RNA polymerases I, II, and III.These enzymes catalyze the transcription of rRNA,(I), mRNA/miRNA (II), and tRNA and 5S rRNA-(III) encoding genes.

RNA polymerase interact with unique cis-active regions of genes, termed promoters, in order to form preinitiation (PICs) capable of initiation. In eukaryotes the process of pol II PIC formation requires, in addition to polymerase, multiple general transcription factors (GTFs), termed TFIIA, B, C, D, F, and H.Eukaryotic PIC formation can occur on accessible promoter either step-wise- by the sequential, ordered interactions of GTFs and RNA polymerase with DNA promoters- or in one step by the recognition of the promoter by a pre-formed GTF RNA polymerase holoenzyme complex.

Transcription exhibits three phases: initiation, elongation, and termination. All are dependent upon distinct DNA cis-element and modulated by distinct trans-acting protein factors.The presence of nucleosomes can occlude the binding of transfactors and the transcription machinery thereby inhibiting transcription.Most eukaryotic RNAs are synthesized as precursors that contain excess sequence which are removed prior to the generation of mature, functional RNA. These processing steps provide additional potential steps for regulation of RNA synthesis.

Eukaryotic mRNA synthesis results in a pre-mRNA precursor that contains extensive amounts of excess RNA (introns) that must be precisely removed by RNA splicing to generate functional, translatable mRNA composed of exonic coding and 5 and 3 noncoding sequences.All steps-form changes in DNA template, sequence, and accessibility in chromatin to RNA stability and translatability-are subject to modulation and hence are potential control sites eukaryotic gene regulation.

Transcription: Synthesis of RNA. (Marks)Transcription is the synthesis of RNA from a DNA template.The enzyme RNA polymerase transcribes genes into a single-stranded RNA.The RNA produced in complementary to one of the strands of DNA, which is known as the template strand. The other DNA strand is the coding, or sense strand..Bacteria contain a single RNA polymerase; eukaryotic cells utilize three different RNA polymerases.

The DNA template is copied in the 3-to5 direction and the RNA transcript is synthesized in the 5-to-3 direction.In contrast to DNA polymerases, RNA polymerase do not require a primer to initiate transcription, nor do they contain error-checking capabilities.Promoter regions, specific sequences in DNA, determine where on the DNA template RNA polymerase binds to initiate transcription.Transcription initiation requires a number of protein factors to allow for efficient RNA polymerase binding to the promoter.

Other DNA sequences, such as promoter-proximal elements and enhancers, affect the rate of transcription initiation through the interactions of DNA-binding proteins with rNA polymerase and other initiation factors.Eucaryotic genes contain exons and introns. Exons specify the coding region of proteins, whereas introns have no coding function.The primary transcript of eukaryotic genes is modified to remove the introns (splicing) before a final, mature RNA is produced.

Protein synthesis and the genetic code. The flow of genetic information follows the sequence DNA RNAProtein.The genetic information in the structural region of a gene is transcribed into an RNA molecule such that the sequence of the latter is complementary to that in the DNA.rRNA, tRNA, and mRNA are directly involved in protein synthesis; miRNAs regulate mRNA function at the level of translation and/or stability.

The information in mRNA is in a tandem array of codons, each of which is three nucleotides long.The mRNA is read continuously from a start codon (AUG) to a termination codon (UAA, UAG, UGA)The open reading frame, or ORF, of the mRNA is the series of codons, each specifying a certain amino acid, that determines the precise amino acid sequence of the protein.Protein synthesis, like DNA and RNA synthesis, follow the 5 to-3 polarity of mRNA and can be divided into three processes; initiation, elongation, and termination.

Mutant proteins arise when single-base substitutions result in codons that specify a different amino acid at a given position, when a stop codon results in a truncated protein, or when base additions or deletions alter the reading frame, so different codons are read.A variety of compounds, including several antibiotics, inhibit protein synthesis by affecting one or more of the steps involved in protein synthesis.

Translation: Synthesis of Protein.(Marks)Translation is the process of translating the sequence of nucleotides in mRNA to an amino acid sequence of a protein.Translation proceeds from the amino terminus to the carboxy terminus, reading the mRNA in the 5 -to-3 direction.Protein synthesis occurs in ribosomes.The mRNA is read in codon, sets of three nucleotides that specify individual amino acids.

AUG, which specifies methionine, is the start codon for all protein synthesis.Specific stop codons ( UAG, UGA, and UAA) signal when the translation of the mRNA is to end.Amino acids are linked covalently to tRNA by the enzyme aminoacyl-tRNA synthetase, creating a charged tRNA.Charged tRNAs base-pair with the codon via the anticodon region of the tRNA.Protein synthesis is divided into three stages: initiation, elongation, and termination.

Multiprotein factors are required for each stage of protein synthesis.Protein fold as they are synthesized.Specific amino acid side chains may be modified after translation by a process known as posttranslational modification.Mechanisms are present within cells to specifically target newly synthesized proteins to different compartment in the cell.

Regulation of gene expression.(Harpers)The genetic constitutions of metazoan somatic cells are nearly all identical.Phenotype (tissue or cell specificity) is dictated by differences in gene expression of this complement of genes.Alterations in gene expression allow a cell to adapt to environmental changes developmental cues, and physiological signals.

Gene expression can be controlled at multiple levels by changes in transcription, RNA processing, localization, and stability or utilization. Gene amplification and rearrangements also influence gene expression.Transcription controls operate at the level of protein-DNA and protein-protein interactions. These intercations display protein domain modularity and high specificity.Several different classes of DNA-binding domains have been identified in transcription factors.

Chromatin and DNA modifications contribute importantly in eukaryotic transcription control by modulating DNA accessibility and specifying recruitment of specific coactivators and corepressors to target genes.miRNA and siRNAs modulate mRNA trans lation and stability; these mechanisms complement transcription controls to regulate gene expression.

Regulation of gene expression. (Marks)Prokaryotic gene expression is regulated primarily at the level of initiation of gene transcription. In general, there is one protein per gene.-Sets of genes that encode proteins with related functions are organized into operons.-Each operon is under the control of a single promoter.-Repressors bind to the promoter to inhibit RNA polymerase binding.-Activators facilitate RNA polymerase bindin to the repressor.

Eukaryotic gene regulation occurs at several levels.-The DNA structural level-chromatin must be remodeled to allow access for RNA polymerase--Transcription is regulated by proteins (specific transcription factors or transactivatorsthat bind to gene regulatory sequences (promoter-proximal elements, response elements, or enhancers).-Transcription factors either enhance or inhibit assembly of the basal transcription complex and RNA polymerase at a promoter.RNA processing (including alternative splicing), transport from the nucleus to the cytoplasm, and translation are also regulated in eukaryotes.

Molecular genetics, Recombinant DNA, & Genomic Technology. (Harpers)A variety of very sensitive techniques can now be applied to the isolation and characterization of genes and to the quantitation of gene products.In DNA cloning, a particular segment of DNA is removed from its normal environment using PCR or one of many restriction endonucleases. This is then ligated into a vector in which the DNA segment can be amplified and produced in abundance.

Cloned DNA can be used as a probe in one of several types of hybridization reactions to detect other related or adjacent pieces of DNA, or it can be used to quantitative gene products such as mRNA.Manipulation of the DNA to change its structure, so-called genetic engineering, is key element in cloning (e.g., the construction of chimeric molecules) and can also be used to study the function of a certain fragment of DNA and to analyze how genes are regulated.

Chimeric DNA molecules are introduced into cells to make transfected cells or into the fertilied oocyte to make transgenic animals.Techniques involving cloned DNA are used to locate genes to specific regions of chromosomes, identify the genes reponsible for diseases, study how faulty gene regulation causes disease, diagnose genetic diseases, and increasingly to treat genetic diseases.

Use of Recombinant DNA Technology in Medicine. (Marks)Techniques for isolating and amplifying genes and studying and manipulating DNA sequences are currently being used in the diagnossis, prevention, and treatment of disease.These techniques require an understanding of the following tools and processes: Restriction enzymes, Cloning vectors, Polymerase Chain Reaction, Dideoxy DNA sequencing, Gel electrophoresis, Nucleic acid hybridization, expression vectors.

Recombinant DNA molecules produced by these techniques can be used as diagnostic probes, in gene therapy, or for the large-scale production of proteins for the treatment of disease.Identified genetic polymorphisms, inherited differences in DNA base sequences between individuals, can be utilized for both diagnosis of disease and the generation of an individuals molecular fingerprint.

Genetic treatment of disease is possible, using either gene therapy or gene ablation techniques. Technical difficulties currently restrict the widespread use of these treatments.Proteomics is the study of proteins expressed by a cell. Differences in protein expression between normal and cancer cells can be used to identify potential targets for future therapy.

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