# DNA Replication

The following proteins are involved in replication

Proteins Involved In Replication

 Protein Function DNA polymerases Deoxyribonucleotides polymerization Helicases Processive unwinding of DNA Topoisomerases Relieve torsional strain that results from helicase-induced unwinding DNA primase Initiates synthesis of RNA primers Single-strand binding proteins Prevent premature reannealing of dsDNA DNA ligase Seals the single strand nick between the nascent chain and Okazaki fragments on lagging Strand or between any two strands

Replication Of Dna Shown Diagramatic

###### Activity of Prokaryotic DNA Polymerases
 Activity DNA Polymerase-I Polymerase-III 5-3 Polymerase activity Present Present 3-5 Exonuclease activity Present Present 5-3 Exonuclease activity Present Present

DNA Pol-II – Participate in DNA repair

 Table: A Comparison of Prokaryotic and Eukaryotic DNA Polymerases E coli Eukaryotic Function I Gap filling following DNA replication, repair, and recombination II DNA proofreading and repair β DNA repair γ Mitochondrial DNA synthesis III ε Processive, leading strand synthesis DnaG α Primazse δ Processive, lagging strand synthesis
1. Telomeres
1. The ends of each chromosome contain structures called telomeres.
2. Telomeres consist of short TG-rich repeats. Human telomeres have a variable number of repeats of the sequence 5'-TTAGGG-3', which can extend for several kilobases.
3. Telomerase, a multisubunit RNA-containing complex related to viral RNA-dependent DNA polymerases (reverse transcriptases), is the enzyme responsible for telomere synthesis and thus for maintaining the length of the telomere.
4. Since telomere shortening has been associated with both malignant transformation and aging, telomerase has become an attractive target for cancer chemotherapy and drug development. Each sister chromatid contains one double-stranded DNA molecule.
5. During interphase, the packing of the DNA molecule is less dense than it is in the condensed chromosome during metaphase.
6. Metaphase chromosomes are nearly completely transcriptionally inactive.
2. #### RNA Synthesis Or Transcription

1. It is very complex process involving one group of polymerase enzyme and a number of proteins
2. No primer is involved in RNA synthesis
3. Only a small part of genome is transcribed
5. Strand of DNA that is transcribed into RNA is called the Template strand
6. Transcription unit is that part of DNA that extends between Promoter and terminator
7. Peptide toxin from mushroom Amanita phylloides ,alpha-amanitine is a specific inhibitor of eukaryotic nucleoplasmic DNA dependent RNA polymerase (RNA polymerase II)
8. Fidelity and frequency of transcription is controlled by proteins bound to certain DNA sequences
9. RNA molecules are processed extensively before they become functional
10. Processing occurs in the nucleus
11. Processing includes removal of introns and splicing of exons,5’ capping,3’ terminal addition, nucleotide modification (methylation, reduction, deamination, and rearranged glycosidic bonds)
3. Alternative Splicing Provides for Different mRNAs
The processing of mRNA molecules is a site for regulation of gene expression. Alternative patterns of mRNA splicing result from tissue-specific adaptive and developmental control mechanisms. Very complex RNA structures are formed during splicing—and that a number of snRNAs and proteins are involved—affords numerous possibilities for a change of this order and for the generation of different mRNAs. The use of alternative termination-cleavage polyadenylation sites also results in mRNA variability.

Fig:
Mechanisms of alternative processing of mRNA precursors. This form of RNA processing involves the selective inclusion or exclusion of exons, the use of alternative 5' donor or 3' acceptor sites, and the use of different polyadenylation sites.

1. Transfer RNA (tRNA) Is Extensively Processed & Modified
The tRNA molecules serve as adapter molecules for the translation of mRNA into protein sequences. The tRNAs contain many modifications of the standard bases A, U, G, and C, including methylation, reduction, deamination, and rearranged glycosidic bonds. Omodification of the tRNA molecules includes nucleotide alkylations and the attachment of the characteristic CpCpAOH terminal at the 3' end of the molecule by the enzyme nucleotidyl transferase.
2. Micro-RNAs Are Derived from Large Primary Transcripts through Specific Nucleolytic Processing
1. The majority of miRNAs are transcribed by RNA pol II into primary transcripts termed pri-miRNAs.
2. pri-miRNAs are 5'-capped and 3'-polyadenylated.
3. Pri-miRNAs are synthesized from transcription units encoding one or several distinct miRNAs; these transcription units are either located independently in the genome or within the intronic DNA of other genes.
4. miRNA-encoding genes must therefore minimally posess a distinct promoter, coding region and polyadenylation/ termination signals.
5. pri-miRNAs have extensive 28 structure, and this intramolecular structure is maintained following processing by the Drosha-DGCR8 nuclease; the portion containing the RNA hairpin is preserved, transported through the nuclear pore and once in the cytoplasm, further processed to a 21 or 22-mer by the dicer nuclease.
6. One of the two strands is selected for loading into the RISC, or RNA induced silencing complex to form a mature, functional miRNA. SiRNAs are produced similarly. Once in the RISC complex, miRNAs can modulate mRNA function

Fig: Biogenesis of miRNAs. miRNA encoding genes are transcribed by RNA pol II into a primary miRNA transcript (pri-miRNA), which is 5'-capped and polyadenylated as is typical of mRNA coding primary transcripts. This pri-miRNA is subjected to processing within the nucleus by the action of the Drosha-DGCR8 nuclease, which trims sequences from both 5' and 3' ends generating the pre-miRNA. This partially processed double-stranded RNA is transported through the nuclear pore by exportin-5. The cytoplasmic pre-miRNA is then trimmed further by the action of the multisubunit nuclease termed Dicer, to form the miRNA duplex. One of the two resulting 21–22 nucleotide-long RNA strands is selected, the duplex unwound, and the selected strand loaded into the RISC complex, thereby generating the mature, functional miRNA.

Extra Edge

1. There are three distinct nuclear DNA-dependent RNA polymerases in mammals: RNA polymerases I, II, and III. These enzymes control the transcriptional function-the transcription of rRNA, mRNA, and small RNA (tRNA /5S rRNA, snRNA) genes, respectively.
2. RNA polymerases interact with unique cis-active regions of genes, termed promoters, in order to form pre initiation complexes (PICs) capable of initiation. In eukaryotes the process of PIC formation is facilitated by multiple general transcription factors (GTFs), TFIIA, B, D, E, F, and H.
3. Transcription exhibits three phases: initiation, elongation, and termination. All are dependent upon distinct DNA cis-elements and can be modulated by distinct trans-acting protein factors
1. Model of Lac Operon

Regulation of prokaryotic gene expression

• In prokaryotes, the genes involved in a metabolic pathway are often present in a linear fashion, called an operon.
• Example:- (i) Lactose operon (Lac operon for regulating lactose metabolism); (ii) Arabinose operon (Ara operon for arabinose metabolism); and (iii) Galactose operon (Gal operon for galactose metabolism).
• Operon contains an operator, a segment of DNA that regulates the activity of the structural genes of the operon.
• If the operator is not bounded by a repressor molecule, RNA polymerase passes over the operator and reaches the protein coding genes and transcribe it into mRNA.
• Ifrepressor molecule is bound to the operator the polymerase is blocked and does not produce the mRNA.
• Thus, as long as the repressor is bound to operator, no proteins are made. When an inducer (activator) molecule is present it binds to the repressor, so that repressor can not bind to operator. Thus, transcription occurs in the presence of an inducer.

Lactose operone or Lac operon

• The lac operon is a region of DNA in the genome of E. coli that contains following genetic elements -
1. Three structural genes:- These code for 3 proteins that are involved in catabolism of lactose. These genes are 'Z' gene (codes for β-galactosidase), 'Y' gene (codes for galactoside permease), and 'A' gene (codes for thiogalactoside transacetylase).
2. Regulatory gene (lac ii) :- It produces repressor protein.
3. A promoter site (P) :- It is the binding site for RNA polymerase. It contains two specific regions-
1. CAP site (Catabolite activator protein binding site).
2. RNA polymerase binding site
4. An operator site (O) :- Repressor binds to this site and blocks transcription
• 3 Structural genes are expressed only when 'O' site is empty (repressor is not bound) and the CAP site is bound by a complex of cAMP and CAP (catabolite gene activator protein).
• 3 situations are possible –
1. When glucose is the only sugar available-
In this case, the lac operon is repressed (turned off). Repression is mediated by the repressor protein binding via helix-turn-helix motif to the operator site. Binding interferes with the progress of RNA polymerase, and blocks transcription of the structural genes. This is an example of negative regulation.
2. When only lactose is available (No glucose).
1. In this case, the lac operon is induced (turned on). A small amount of lactose is converted to an isomer,  allolactose. This compound is an inducer that binds to the repressor protein, changing its configuration so that it can no longer bind to the operator.
2. In absence of glucose, adenylyl cyclase is active and sufficient quantitive of cAMP are made and bind to the CAP-binding site, causing RNA polymerase to more efficiently initiate transcription at the promoter site. This is an ego of positive regulation.
3. When both glucose and lactose are available
1. In this case transcription of lac operon is negligible, even if lactose is present at a higher concentration. Adenylyl cyclase is deactivated in the presence of glucose-a process known as catabolite repression, So no cAMP-CAP complex forms and the CAP binding site remains empty.
2. RNA polymerase is unable to effectively initiate the transcription, even though the repressor may not be bound to the operator region.

Several Motifs Mediate the Binding of Regulatory Proteins to DNA

• The specificity involved in the control of transcription requires that regulatory proteins bind with high affinity and specificity to the correct region of DNA. Three unique motifs—the helix-turn-helix, the zinc finger, and the leucine zipper—account for many of these specific protein-DNA interactions. Examples of proteins containing these motifs are given in Table below
1. Comparison of the binding activities of the proteins that contain these motifs leads to several important generalizations.
1. Binding must be of high affinity to the specific site and of low affinity to other DNA.
2. Small regions of the protein make direct contact with DNA; the rest of the protein, in addition to providing the trans-activation domains, may be involved in the dimerization of monomers of the binding protein, may provide a contact surface for the formation of heterodimers, may provide one or more ligand-binding sites, or may provide surfaces for interaction with coactivators or corepressors.
3. The protein-DNA interactions are maintained by hydrogen bonds, ionic interactions and van der Waals forces.
4. The motifs found in these proteins are unique; their presence in a protein of unknown function suggests that the protein may bind to DNA.
5. Proteins with the helix-turn-helix or leucine zipper motifs form dimers, and their respective DNA binding sites are symmetric palindromes. In proteins with the zinc finger motif, the binding site is repeated two to nine times. These features allow for cooperative interactions between binding sites and enhance the degree and affinity of binding.
 Table- Examples of Transcription Regulatory Proteins That Contain the Various Binding Motifs

 Binding Motif Organism Regulatory Protein Helix-turn-helix E coli Phage Mammals lac repressor CAP λcI, cro, and tryptophan and 434 repressors Homeobox proteins Pit-1, Oct1, Oct2 Zinc finger E coli Yeast Drosophila Xenopus Mammals Gene 32 protein Gal4 Serendipity, Hunchback TFIIIA Steroid receptor family, Sp1 Leucine zipper Yeast Mammals GCN4 C/EBP, fos, Jun, Fra-1, CRE binding protein, c-myc, n-myc, I-myc

1. Accelerated Aging and the cell nucleus
1. Hutchinson-Gilford progeria syndrome (HGPS)- is a very rare condition where patients experience a rapid rate of aging begin­ning at birth. It is detected early in life with limited growth, loss of hair, distinctive facial characteristics, small fragile bodies, wrinkled skin, atherosclerosis, and cardiovascular problems, all of which are common in the elderly. Patients do not, however, have symptoms of neurodegen­erative diseases.
2. They rarely live past the early teens.
3. The aging process in these individuals is 6 to 8 times the normal rate of aging.
4. A genetic change from normal has been identified in patients with HGPS. The nuclear envelope of patients appears distorted. Lamina A, a structural protein in nuclei, is involved in nuclear syn­thesis of DNA and RNA. It is formed from a precursor, attached to the nuclear membrane, by an enzyme-catalyzed reaction.
2. Protein Synthesis (Translation)
1. The letters A, G, T and C correspond to the nucleotides found in DNA, they are arranged in 3 letter code word called codon. Collection of these codons is called Genetic Code.
2. The genetic code is DEGENERATE, UNAMBIGUOUS, NONOVERLAPPING, WITHOUT PUNCTUATION, & UNIVERSAL.
3. In prokaryotes there is a linear correspondence between the gene, the mRNA transcribed from the gene, and the polypeptide product
4. In eukaryotes the primary transcript is hnRNA (or pre-mRNA) which is larger than mature mRNA. hnRNA contains exons which combine to form mRNA and introns which are removed during process known as splicing.
5. The nucleotide sequence of mRNA contains series of codons that specifies the amino acid sequence of encoded protein
6. 3 codons do not code for specific amino acids; these have been termed NONSENSE CODONS: UAA, UAG, and UGA.
7. AUG which serves as the initiator codon in mammalian cells also serves as codon for Methionine.
8. At least one species of tRNA exists for each of the 20 amino acids. These molecules have extraordinarily similar function and 3-D structure
9. The adapter function of tRNA requires the charging of each specific tRNA with its specific amino acid .the process of recognition and attachment (charging) is carried out in 2 steps by one enzyme for each of the 20 amino acids called aminoacyl tRNA synthetase. For each amino acid to bind with its specific tRNA, two ATPs are required.
10. The anticodon arm of tRNA consists of seven nucleotides ,and it recognizes 3 letter codon in mRNA. The sequence in is read from 3’ to 5’ in anticodon whereas genetic code is read in 5’ to 3’
11. The degeneracy of genetic code resides mostly in last nucleotide of the triplet, suggesting that the base pairing between this last nucleotide and the corresponding nucleotide of anticodon is not strictly by Watson Crick rule. This is called Wobble.
12. Protein synthesis can be described in 3 phases: initiation ,elongation, and termination
13. A total of 4 high energy bonds are utilized for formation of one peptide bond between two amino acids
14. There are two control points for protein synthesis: eIF-2 and eIF-4E
15. Termination of protein synthesis occurs when a nonsense codon is recognized
16. Many antibiotics work because they inhibit protein synthesis in bacteria: tetracycline, lincomycin, erythromycin and chloramphenicol
17. Diphtheria toxin catalyses the ADP-ribosyl tion of eEF2in mammalian cell. this modification inactivates eEF2and thereby specifically inhibits mammalian protein synthesis
18. Ricin an extremely toxic molecule, isolated from castor bean, inactivates eukaryotic 28SrRNAby providing N glycosidic cleavage or removal of single adenine
###### Eukaryotic Translation Shown Diagrammatically

Extra Edge
1. The mRNA is read continuously from a start codon (AUG) to a termination codon (UAA, UAG, UGA).
2. The open reading frame of the mRNA is the series of codons, each specifying a certain amino acid, that determines the precise amino acid sequence of the protein
3. Protein synthesis, like DNA and RNA synthesis, follows a 5’ to 3’ polarity 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 codons results in a truncated protein, or when base additions or deletions alter the reading frame, so different codons are read.
4. Alteration in gene expression allow a cell to adapt to environmental changes.
5. 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.
6. Transcription controls operate at the levels of protein-DNA and protein-protein interactions. These interactions display protein domain modularity and high specificity.
7. In DNA cloning, a particular segment of DNA is removed from its normal environment using restriction endonucleases. This is then inserted into a vector in which the DNA segment can amplified and produced in abundance.
8. Cloned DNA can be used as a probe in one of several of hybridization reactions to detect other related adjacent pieces of DNA.
9. Chimeric DNA molecules are introduced into cells to make transected cells or into the fertilized oocyte to make transgenic animals.

Wobble hypothesis

Degeneracy (Redundancy) of codes can be explained by wobble hypothesis for codon-anticodon interaction. Each codon base pairs with anticodon of tRNA in antiparalled fashion. First two bases of codons are the same whereas the third is different, “wobble”. First two bases of codon pair with last two bases of anticodon with normal watson –crick base pairing. Base pairing of 3rd base of codon (at 31 end) with 1st base of anticodon (at 51 end at tRNA) does not strictly follow Watson – crick base pairing rule.

Suppressor Mutations Can Counteract Some of the Effects of Missense, Nonsense, & Frameshift Mutations

In prokaryotic and lower eukaryotic organisms, abnormally functioning tRNA molecules have been discovered that are themselves the results of mutations. Some of these abnormal tRNA molecules are capable of binding to and decoding altered codons, thereby suppressing the effects of mutations in distinct mutated mRNA encoding structural genes. These suppressor tRNA molecules, usually formed as the result of alterations in their anticodon regions, are capable of suppressing certain missense mutations, nonsense mutations, and frameshift mutations.

Gene therapy : The goal of gene therapy is to replace the defective, disease causing genes by normal functioning copies in the afflicted humans. Three approaches for gene therapy are: Gene replacement, Gene augmentation (most commonly used). Gene therapy may be somatic cell gene therapy or germline gene therapy.

Gene Regulation in Eukaryotes :-

• It is more complex. Operons are not present, but coordinated regulation of the transcription of genes located on different chromosomes can be achieved through the binding of trans-acting proteins to cis-acting elements.
• In multicellular organisms, hormones can cause coordinated regulation, either through the binding of a protein that is activated in response to a second messenger or through the binding of the hormone receptor-hormone complex itself to the DNA. In each case, binding to the DNA is mediated through structured motifs such as the zinc-finger motifs . Co-and Post transcriptional regulation is also seen and it includes splice-site choice, poly-A-site choice, m-RNA editing and variation in mRNA stability as seen with transferring receptor synthesis and with RNA interference.
• Regulation at the transcriptional level can be caused by phosphorylation of eIF-2.
• Gene expression in eukaryotes is also influenced by availability of DNA to transcriptional apparatus, the amount of DNA, and the arrangement of the DNA.
1. ###### Important Terms In Recombinant DNA Technology
1. Chimeric DNA: molecules containing both human and bacterial DNA in sequence independent manner.
2. Restriction enzyme: They cut DNA at specific site; they are called restriction because their presence in bacteria restricts the growth of virus in them
3. Clone: a large population of identical cells that arise from common ancestor.
4. Plasmids: Is small circular DNA that confers antibiotic resistance to the bacteria. It grows separately from host DNA and replicates independently from it. It can accept foreign DNA of 6-10 Kb long.
5. Phages: have linear DNA to which foreign DNA can be inserted at several sites, up to 10-20Kb long.
6. Cosmids: can carry foreign DNA up to 35-50Kb long.
7. Genomic library: prepared from total DNA of a cell line. Entire genome of an organism is packaged into vectors using various RE’s.
8. cDNA library: comprises complementary DNA’s of population of mRNA in a tissue
9. Expression Vector: a vector in which the protein coded by the gene introduced by recombinant technology is actually synthesized.
10. Probes: search library for specific genes or cDNA molecules. They are generally pieces of DNA or RNA labeled with 32-P containing nucleotide or fluorescently labeled nucleotides.
11. Blot Transfer technique: These procedures are useful in determining how many copies of a gene are present in a given tissue or if there are any gross alteration in a gene of a given tissue.
1. Southern Blot Transfer is for detecting DNA
2. Northern Blot Transfer is for detecting RNA
3. Western Blot Transfer is for detecting Protein (AIIMS Nov 09)
4. South-West Blot Transfer is for detecting Protein-DNA interaction
12. PCR: Polymerase Chain Reaction amplifies a target sequence of DNA. It is sensitive, selective and rapid. A heat stable DNA polymerase from Thermus acquaticus known as Taq Polymerase is used for the procedure.
13. RT-PCR: In this mRNA of a given tissue are amplified, but first the RNA is used to generate cDNA copies using retroviral reverse transcriptase.
14. In situ hybridization: In this a radioactive probe is added to metaphase spread of chromosome on a glass slide. The exact area of hybridization is localized by layering photographic emulsion over the slide. This technique places the gene at a given location on a given band or region on chromosome.
15. Transgenic animals: their germ lines have been altered. Genes are injected into fertilized ovum, hence are incorporated into both somatic and germ cells of an organism.
16. Gene Knockout animals: created by making a mutation that totally disrupts the function of a gene.
2. Important Terms In Genetics
1. ARS: Autonomously replicating sequence; the origin of replication in yeast.
2. Autoradiography: The detection of radioactive mole­cules (eg, DNA, RNA, protein) by visualization of their effects on photographic film.
3. Bacteriophage: A virus that infects a bacterium.
4. Blunt-ended DNA: Two strands of a DNA duplex having ends that are flush with each other.
5. cDNA: A single-stranded DNA molecule that is com­plementary to an mRNA molecule and is synthe­sized from it by the action of reverse transcriptase.
6. Chimeric molecule: A molecule (eg, DNA, RNA, protein) containing sequences derived from two different species.
7. Clone: A large number of organisms, cells or mole­cules that are identical with a single parental organ­ism cell or molecule.
8. Cosmid: A plasmid into which the DNA sequences from bacteriophage lambda that are necessary for the packaging of DNA (cos sites) have been inserted; this permits the plasmid DNA to be packaged in vitro.
9. Endonuclease: An enzyme that cleaves internal bonds in DNA or RNA.
10. Excinuclease: The excision nuclease (endonuclease) involved in nucleotide excision repair of DNA.
11. Exon: The sequence of a gene that is represented (expressed) as mRNA.
12. Exonuclease: An enzyme that cleaves nucleotides from either the 3' or 5' ends of DNA or RNA.
13. Fingerprinting: The use of RFLPs or repeat sequence DNA to establish a unique pattern of DNA frag­ments for an individual.
14. Footprinting: DNA with protein bound is resistant to digestion by DNase enzymes. When a sequencing reaction is performed using such DNA, a protected area, representing the "footprint" of the bound pro­tein, will be detected.
15. Hairpin: A double-helical stretch formed by base pair­ing between neighboring complementary sequences of a single strand of DNA or RNA.
16. Hybridization: The specific reassociation of comple­mentary strands of nucleic acids (DNA with DNA, DNA with RNA, or RNA with RNA).
17. Insert: An additional length of base pairs in DNA, generally introduced by the techniques of recombi­nant DNA technology.
18. Intron: The sequence of a gene that is transcribed but excised before translation.
19. Library: A collection of cloned fragments that repre­sents the entire genome. Libraries may be either genomic DNA (in which both introns and exons are represented) or cDNA (in which only exons are represented).
20. Ligation: The enzyme-catalyzed joining in phosphodi­ester linkage of two stretches of DNA or RNA into one; the respective enzymes are DNA and RNA ligases.
21. Lines:  long interspersed repeat sequences
22. Microsatellite polymorphism: Heterozygosity of a certain microsatellite repeat in an individual.
23. Microsatellite repeat sequences: Dispersed or group repeat sequences of 2-5 bp repeated up to 50 times. May occur at 50-100 thousand locations in the genome.
24. miRNAs: Micro RNAs, 21-25 nucleotide long RNA species derived from RNA polymerase II transcrip­tion units, 500-1500 bp in length via RNA process­ing. These RNAs, recently discovered, are thought to play crucial roles in gene regulation.
25. Nick translation: A technique for labeling DNA based on the ability of the DNA polymerase from E coli to degrade a strand of DNA that has been nicked and then to re synthesize the strand; if a radioactive nucleoside triphosphate is employed, the rebuilt strand becomes labeled and can be used as a radioactive probe.
26. Northern blot: A method for transferring RNA from an agarose gel to a nitrocellulose filter, on which the RNA can be detected by a suitable probe.
27. Oligonucleotide: A short, defined sequence of nucle­otides joined together in the typical phosphodiester linkage.
28. Ori: The origin of DNA replication.
29. PAC: A high-capacity (70-95 kb) cloning vector based upon the lytic E coli bacteriophage PI that replicates in bacteria as an extrachromosomal element.
30. Palindrome: A sequence of duplex DNA that is the same when the two strands are read in opposite direction.
31. Plasmid: A small, extrachromosomal, circular DNA mole­cule that replicates independently of the host DNA.
32. Polymerase chain reaction (PCR): An enzymatic method for the repeated copying (and thus amplifi­    cation) of the two strands of DNA that make up a particular gene sequence.
33. Primosome: The mobile complex of helicase and primase that is involved in DNA replication.
34. Probe: A molecule used to detect the presence of a specific fragment of DNA or RNA in, for instance, a bacterial colony that is formed from a genetic library or during analysis by blot transfer tech­niques; common probes are cDNA molecules, syn­thetic oligodeoxynucleotides of defined sequence, or antibodies to specific proteins.
35. Proteome: The entire collection of expressed proteins in an organism.
36. Pseudogene: An inactive segment of DNA arising by mutation of a parental active gene.
37. Recombinant DNA: The altered DNA that result from the insertion of a sequence of deoxy nucle-o tides not previously present into an existing mole-cule of DNA by enzymatic or chemical means.
38. Restriction enzyme: An endodeoxynuclease that causes cleavage of both strands of DNA at highly specific sites dictated by the base sequence, known as restriction site.
39. Reverse transcription: RNA-directed synthesis of DNA, catalyzed by reverse transcriptase.
40. RT-PCR: A method used to quantitate mRNA levels that relies upon a first step of cDNA copying of mRNAs catalyzed by reverse transcriptase prior to PCR amplification and quantitation.
41. Signal: The end product observed when a specific sequence of DNA or RNA is detected by autoradiography or some other method. Hybridization with a complementary radioactive polynucleotide (eg, by Southern or Northern blotting) is commonly used to generate the signal.
42. Sines: Short interspersed repeat sequences.
43. SiRNAs: Silencing RNAs, 21-25 nt in length generated by selective nucleotide degradation of double­ stranded RNAs of cellular or viral origin. SiRNAs anneal to various specific sites within target in RNAs leading to mRNA degradation, hence gene "knockdown."
44. SNP: Single nucleotide polymorphism. Refers to the fact that single nucleotide genetic variation in genome sequence exists at discrete loci throughout the chromosomes. Measurement of allelic SNP dif­ferences is useful for gene mapping studies.
45. snRNA: Small nuclear RNA. This family of RNAs is best known for its role in mRNA processing.
46. Southern blot: A method for transferring DNA from an agarose gel to nitrocellulose filter, on which the DNA can be detected by a suitable probe (e.g., com­plementary DNA or RNA).
47. Southwestern blot: A method for detecting protein­ DNA interactions by applying a labeled DNA probe to a transfer membrane that contains a rena­tured protein.
48. Spliceosome: The macromolecular complex responsi­ble for precursor mRNA splicing. The splice some consists of at least five small nuclear RNAs (snRNA; Ul, U2, U4, U5, and U6) and many proteins.
49. Splicing: The removal of introns from RNA accom­panied by the joining of its exons.
1. Sticky-ended DNA: Complementary single strands of DNA that protrude from opposite ends of a DNA duplex
2. or from the ends of different duplex mole­cules (see also Blunt-ended DNA, above).
3. Tandem: Used to describe multiple copies of the same sequence (e.g. DNA) that lie adjacent to one another.
4. Terminal transferase: An enzyme that adds nucle­otides of one type (e.g. deoxyadenonucleotidyl resi­dues) to the 3' end of DNA strands.
5. Transcription: Template DNA-directed nucleic acids; typically DNA-directed RNA.
6. Transcriptase: The entire collection of expressed mRNAs in an organism.
7. Transgenic: Describing the DNA into germ cells by nucleus of the ovum.
8. Translation: Synthesis of protein using mRNA as template.
9. Vector: A plasmid or bacteriophage into which for­eign DNA can be introduced for the purposes of cloning.
10. Western blot: A method for transferring protein to a nitrocellulose filter, on which the protein can be de­tected by a suitable probe (e.g., an antibody).