molecular biology 1
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molecular biology 1 - Leaderboard
molecular biology 1 - Details
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| What's the hereditary basis of every living organism ? | Its their genome |
| What are restriction endonuclease? | An enzyme that recognizes specific short sequences of DNA and cleaves the duplex (sometimes at the target site, sometimes elsewhere, depending on type). |
| What does the genome include ? | DNA, plasmids (bacteria) , organelles DNA |
| What are nucleases and phosphatases? | Nucleases hydrolyze an ester bond within a phosphodiester bond,Phosphatases hydrolyze the ester bond in a phosphomonoester bond. |
| What are chromosomes ? | A discrete unit of the genome earring many genes, each one consists of DNA and proteins. |
| What is the difference between endonuclease and exonuclease? | Endonuclease – Nuclease that cleaves phosphoester bonds within a nucleic acid chain (It may be specific for RNA or for single-stranded or double-stranded DNA). exonuclease – Nuclease that cleaves phosphoester bonds one at a time from the end of a polynucleotide chain (It may be specific for either the 5′ or 3′ end of DNA or RNA). |
| What's a gene? | A sequence of DNA that encodes an RNA and in protein coding genes. |
| What can restriction endonuclease be used for? | It can be used to cleave DNA into defined fragments. |
| What is transformation ? | Its when genetic properties can be transferred from one bacteria strain to another, and its the first support that DNA is the genetic material of bacteria. |
| What can be generated using the overlaps of fragments? | A map can be generated by using the overlaps between the fragments generated by different restriction enzymes. |
| What is the transforming principle ? | DNA is taken up by a bacterium it changes the properties of the recipient . |
| What are cloning vectors? | DNA ( derived from a plasmid, bacteriophage genome) that can be used to propagate an incorporated DNA sequence in a host cell.Vectors contain selectable markers and replication origins to allow identification and maintenance of the vector in the host. |
| What is transfection? | Its the inquisition of new genetic markers , by incorporating added DNA |
| What cloning a DNA fragment require ? | A fragment of DNA requires a specially engineered vector. |
| What does a nucleoside consist of? what do they link onto in DNA and RNA? | Purines or pyrimidines, and they link to 1' on DNA and 2' on RNA carbon of the pentose sugar. |
| What is recombinant DNA ? | A DNA molecule that has been created by joining together two or more molecules from different sources. |
| What does subclone mean? | The process of breaking a cloned fragment into smaller fragments for further cloning. |
| What is a MCS? | Multiple cloning site is a sequence of DNA containing a series of tandem restriction endonuclease sites used in cloning vectors for creating recombinant molecules. |
| What are cloning vectors divided into? | Can Be Specialized for Different Purposes. |
| How can DNA be introduced into different target cells? | With these methods |
| What's a probe? | A radioactive nucleic acid, DNA or RNA, used to identify a complementary fragment. |
| What's autoradiography (nucleic acid detection) | A method of capturing an image of radioactive materials on film. |
| What's in situ hybridization? | Hybridization of a probe to intact tissue to locate its complementary strand by autoradiography. |
| What are the DNA sequencing steps? | Gel electrophoresis separates DNA fragments by size, using an electric current to cause the DNA to migrate toward a positive charge. |
| What is the PCR and RT-PCR tesuniqes? | Polymerase chain reaction permits the exponential amplification of a desired sequence, using primers that anneal to the sequence of interest,RT-PCR uses reverse transcriptase to convert RNA to DNA for use in a PCR reaction. |
| What is southern blotting ? | Involves the transfer of DNA from a gel to a membrane, followed by detection of specific sequences by hybridization with a labeled probe. |
| What is northern blotting ? | Is similar to Southern blotting, but involves the transfer of RNA from a gel to a membrane. |
| What is western blotting? | Entails separation of proteins on a sodium dodecyl sulfate (SDS) gel, transfer to a nitrocellulose membrane, and detection of proteins of interest using antibodies. |
| What's an epitope tag? | A short peptide sequence that encodes a recognition site (“epitope”) for an antibody, typically fused to a protein of interest for detection or purification by the antibody. |
| What are microarrays? | They comprise known DNA sequences spotted or synthesized on a small chip. |
| What's next generation sequencing (NGS), what does it refer to? | Next-generation sequencing is based on the ability to sequence, in parallel, millions of DNA fragments, Next generation sequencing (NGS) refers to large-scale DNA sequencing technology that allows for querying - the entire genome (whole genome), - the exons within all known genes (whole exome), - or only exons of selected genes (target panel). |
| How is genome-wide transcription analysis is performed? | Is performed using labeled cDNA from experimental samples hybridized to a microarray containing sequences from all ORFs of the organism being used. |
| What are SNP arrays? | They permit genome-wide genotyping of single-nucleotide polymorphisms. |
| What is RNA sequencing? | Uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment. |
| What does transgenics mean? | Organisms created by introducing DNA prepared in test tubes into the germline. The DNA may be inserted into the genome or exist in an extrachromosomal structure. |
| Embryonic stem (ES)? what do they contribute to? | Cells that are injected into a mouse blastocyst generate descendant cells that become part of a chimeric adult mouse. -When the ES cells contribute to the germline, the next generation of mice may be derived from the ES cell. -Genes can be added to the mouse germline by transfecting them into ES cells before the cells are added to the blastocyst. |
| How can an endogenous gene be replaced? | Be replaced by a transfected gene using homologous recombination. |
| How can a successful homologous recombination be detected? | By using two selectable markers, one of which is incorporated with the integrated gene, the other of which is lost when recombination occurs. |
| What are knockouts and knock-ins in the cre//ox ? | Knockout – A process in which a gene function is eliminated, usually by replacing most of the coding sequence with a selectable marker in vitro and transferring the altered gene to the genome by homologous recombination. knock-in – A process similar to a knockout, but more subtle mutations are made. |
| Array comparative genome hybridization? | (array-CGH) allows the detection of copy number changes in any DNA sequence compared between two samples. |
| What has the Introduction of NGS has resulted in ? | -a dramatic increase in speed and content of sequencing -at a fraction of the cost |
| When is replication initiated? | At the bacterial origin when a cell passes a critical threshold of size. |
| What is a replicon? | A unit of the genome in which DNA is replicated. Each contains an origin for initiation of replication. |
| What is the origin? | A sequence of DNA at which replication is initiated. |
| What is the terminus? | A segment of DNA at which replication ends. |
| What is single-copyreplicationcontrol and multicopyreplicationcontrol? | Single-copyreplicationcontrol control system in which there is only one copy of a replicon per unit bacterium,– The bacterial chromosome and some plasmids have this type of regulation.multicopyreplicationcontrol–Occurswhen the control system allows the plasmid to exist in more than one copy per individual bacterial cell. |
| What is semiconservative replication? | Replication accomplished by separation of the strands of a parental duplex, with each strand then acting as a template for synthesis of a complementary strand. |
| What appears as a bubble within nonreplicated DNA? | A replicated region |
| What is the replication fork? | Is initiated at the origin and then moves sequentially along DNA. |
| What are the types of replication? | Unidirectional replication when a single replication fork is created at an origin. bidirectional replication when an origin creates two replication forks that move in opposite. |
| How is does the Bacterial Genome replicate? | Bacterial replicons are usually circles that replicate bidirectionally from a single origin. The origin of E. coli, oriC, is 245 bp in length. |
| How does Methylation of the Bacterial Origin Regulates Initiation? | OriC contains binding sites for DnaA: dnaA-boxes. • oriC also contains 11 GATC/CTAG repeats that are methylated on adenine on both strands. |
| What does Replication generates? | Hemimethylated DNA, which cannot initiate replication. • There is a 13-minute delay before the GATC/CTAG repeats are remethylated. |
| What does Initiation at oriC require? | Requires the sequential assembly of a large protein complex on the membrane. • oriC must be fully methylated. • DnaA-ATP (a licensing factor) binds to short repeated sequences and forms an oligomeric complex that melts DNA. |
| What does Creating the Replication Forks at the Origin oriC involve ? | Six DnaC monomers bind each hexamer of DnaB, and this complex binds to the origin. • A hexamer of DnaB (a helicase) forms the replication fork. Gyrase and single-strand binding proteins (SSB) are also required. • A short region of A-T–rich DNA is melted. • DnaG primase is bound to the helicase complex and creates the replication forks. |
| What are the Mechanisms Exist to Prevent Premature Reinitiation of Replication? | SeqA binds to hemimethylated DNA and is required for delaying rereplication. • SeqA may interact with DnaA. • As the origins are hemimethylated they bind to the cell membrane and may be unavailable to methylases. • The dat locus contains DnaA-binding sites that titrate availability of DnaA protein. • Hda protein is recruited to the replication origin to convert DnaA-ATP to DnaA-ADP. |
| Do Archaeal Chromosomes Contain Multiple Replicons? | Some archaea have multiple replication origins. • These origins are bound by homologs of eukaryotic replication initiation factors. |
| Do Eukaryotic Chromosome Contains Many Replicons? | A chromosome is divided into many replicons. • The progression into S phase is tightly controlled. |
| How large are the replicons in eukaryotic cell? and how are they activated? | Eukaryotic replicons are 40 to 100 kb in length. • Individual replicons are activated at characteristic times during S phase. • Regional activation patterns suggest that replicons near one another are activated at the same time. |
| What are the Origins that Can Be Isolated in Yeast? | Origins in S. cerevisiae are short A-T sequences that have an essential 11-bp sequence. • The ORC is a complex of six proteins that binds to an ARS. |
| What is the domain of the Replication Origins Can Be Isolated in Yeast? where can related ORC be found? | The conserved 11-bp sequence of A-T base pairs in the yeast ARS element that comprises the replication origin.Related ORC complexes are found in multicellular eukaryotes. |
| What are Licensing factor ? | It is necessary for initiation of replication at each origin. • Licensing factor is present in the nucleus prior to replication, but is removed, inactivated, or destroyed by replication. |
| What are the Licensing Factor that Bind to ORC? | The ORC is a protein complex that is associated with yeast origins throughout the cell cycle. • Cdc6 protein is an unstable protein that is synthesized only in G1. • Cdc6 binds to ORC and allows MCM proteins to bind. • Cdt1 facilitates MCM loading on origins. •When replication is initiated, Cdc6 and Cdt1 are displaced. The degradation of Cdc6 prevents reinitiation. |
| What are prereplication complex and postreplication complex? | Prereplication complex – A protein-DNA complex at the origin in S. cerevisiae that is required for DNA replication. The complex contains the ORC complex, Cdc6, and the MCM proteins.postreplication complex – A protein-DNA complex in S. cerevisiae that consists of the ORC complex bound to the origin. |
| What's a checkpoint? | A biochemical control mechanism that prevents the cell from progressing from one stage to the next unless specific goals and requirements have been met. |
| What's mismatch repair (MMR) ? | A type of repair that corrects mispaired bases, typically immediately following replication. -The process preferentially corrects the sequence of the daughter strand by distinguishing the daughter strand and parental strand, sometimes on the basis of their states of methylation. |
| What's photoreactivation (Direct Repair) ? | A repair mechanism that uses a white light-dependent enzyme to split cyclobutane pyrimidine dimers formed by ultraviolet light. |
| What's excision repair ? | A type of repair system in which one strand of DNA is directly excised and then replaced by resynthesis using the complementary strand as template. |
| What's base excision repair (BER) ? | A pathway of excision repair that recognizes damage to single bases, such as deaminiation or alkylation, and either repairs the base alone (short-patch repair) or replaces 2–10 nucleotides (long-patch repair). |
| What's nucleotide excision repair (NER) ? | An excision repair pathway that recognizes bulky lesions in DNA (such as UV-induced pyrimidine dimers). -NER is divided into two major subpathways: transcription-coupled repair (TC-NER), which repairs damaged in the transcribed strand of active genes; and global genome repair (GG-NER), which repairs damage anywhere in the genome. |
| How does the DNA repair system correct damaged DNA? | -Repair systems recognize DNA sequences that do not conform to standard base pairs. -Excision systems remove one strand of DNA at the site of damage and then replace it. |
| What are the Types of damages that trigger repair system ? | -Single base changes. -Structural distortions/bulky lesions. -Strand breaks. |
| What happens in single base changes? | Corrected by either replacing U with C or corrected by removing A or G. |
| What happens in Structural distortions? | -Introduction of covalent links between bases on one strand of DNA or between bases of opposite strands. -Photoreactivation is a nonmutagenic repair system that acts specifically on pyrimidine dimers. |
| What happens when a DNA strand breaks? | -Can occur in one or both strands. -A single –stranded break, or nick, can be directly ligated. -DSBs are major damage, if unrepaired, can result in extensive loss of DNA. |
| What's the Excision Repair Systems in E. coli? | -The Uvr system makes incisions ~12 bases apart on both sides of damaged DNA, removes the DNA between them (excision), and resynthesizes new DNA. -Transcribed genes are preferentially repaired when DNA damage occurs. |
| What's Xeroderma pigmentosum (XP) ? | It is a human disease caused by mutations in any one of several nucleotide excision repair genes. -Numerous proteins, including XP products and the transcription factor TFIIH, are involved in eukaryotic nucleotide excision repair. |
| What are Eukaryotic Nucleotide Excision Repair Pathways? | -Global genome repair recognizes damage anywhere in the genome. -Transcriptionally active genes are preferentially repaired via transcription-coupled repair. -Global genome repair and transcription-coupled repair differ in their mechanisms of damage recognition (XPC vs. RNA polymerase II). |
| What's the Eukaryotic Nucleotide Excision Repair Pathways link to a complex of repair enzymes and what's the mutation that occurs? | TFIIH provides the link to a complex of repair enzymes. Mutations in the XPD component of TFIIH cause three types of human diseases. |
| What's Base Excision Repair Systems Require Glycosylases? | -Base excision repair is triggered by directly removing a damaged base from DNA. -Base removal triggers the removal and replacement of a stretch of polynucleotides. -The nature of the base removal reaction determines which of two pathways for excision repair is activated. -The polδ/ε pathway replaces a long polynucleotide stretch; the polβ pathway replaces a short stretch. |
| Uracil and alkylated bases are recognized by what enzyme?what does this enzyme do? | -bases are recognized by glycosylases and removed directly from DNA. -Glycosylases and photolyase (a lyase) act by flipping the base out of the double helix, where, depending on the reaction, it is either removed or modified and returned to the helix. |
| What are Error-Prone Repair and Translesion Synthesis? | -Damaged DNA that has not been repaired causes DNA polymerase III to stall during replication. -DNA polymerase V (coded by umuCD) or DNA polymerase IV (coded by dinB) can synthesize a complement to the damaged strand. -The DNA synthesized by repair DNA polymerases often has errors in its sequence (error-prone synthesis). |
| What's a mutator? what gene encodes for that? | -A mutation or a mutated gene that increases the basal level of mutation. Such genes often code for proteins that are involved in repairing damaged DNA. -The mut genes code for a mismatch repair system that deals with mismatched base pairs. |
| How is the Direction of Mismatch Repair Controlled? | -There is a bias in the selection of which strand to replace at mismatches. -The strand lacking methylation at a hemimethylated GATCCTAG is usually replaced. -The mismatch repair system is used to remove errors in a newly synthesized strand of DNA. At G-T and C-T mismatches, the T is preferentially removed. |
| How is The mismatch repair system is used ? | Eukaryotic MutS/L systems repair mismatches and insertion/deletion loops |
| What's The recombination-repair system? | -The rec genes of E. coli code for the principal recombination-repair system. -The recombination-repair system functions when replication leaves a gap in a newly synthesized strand that is opposite a damaged sequence. |
| How does Recombination-Repair Systems in E. coli happen? | -The single strand of another duplex is used to replace the gap (single-strand exchange). -The damaged sequence is then removed and resynthesized. |
| How does Recombination occur to Recover from Replication Errors? | A replication fork may stall when it encounters a damaged site or a nick in DNA. A stalled fork may reverse by pairing between the two newly synthesized strands. |
| How is Recombination-Repair used for Double-Strand Breaks in Eukaryotes? | -The yeast RAD mutations, identified by radiation-sensitive phenotypes, are in genes that code for repair systems. -The RAD52 group of genes is required for recombination repair. -The MRX (yeast) or MRN (mammals) complex is required to form a single-stranded region at each DNA end. |
| How does a nucleoprotein filament on the single-stranded region formed? | By the The RecA homolog Rad51 assisted by Rad52 and Rad55/57 (involved in homology search and strand invasion). |
| How are Nonhomologous End-Joining and how do they Repair Double-Strand Breaks? | -Repair of double-strand breaks when homologous sequence is not available occurs through a nonhomologous end-joining (NHEJ) reaction. -The NHEJ pathway can ligate blunt ends of duplex DNA. -Mutations in double-strand break repair pathways cause human diseases. |
| How does DNA Repair in Eukaryotes Occur in the Context of Chromatin? | -Both histone modification and chromatin remodeling are essential for repair of DNA damage in chromatin. -H2A phosphorylation (y-H2AX) is a conserved double-strand break-dependent modification that recruits chromatin modifying activities and facilitates assembly of repair factors. |
| What does different patterns of histones distinguish, and what is required to reset chromatin? | -Different patterns of histone modifications may distinguish stages of repair or different pathways of repair. -Remodelers and chaperones are required to reset chromatin structure after completion of repair. |
| How does RecA Trigger the SOS System? | -Damage to DNA causes RecA to trigger the SOS response, which consists of genes coding for many repair enzymes. -RecA activates the autocleavage activity of LexA. -LexA represses the SOS system; its autocleavage activates those genes. |
| What is a transposon (transposable element) ? | A DNA sequence able to insert itself (or a copy of itself) at a new location in the genome without having any sequence relationship with the target locus. |
| What are retroviruses? | An RNA virus with the ability to convert its sequence into DNA by reverse transcription. |
| What is a retrotransposon (retroposon) ? | -A transposon that mobilizes via an RNA form; the DNA element is transcribed into RNA, and then reverse-transcribed into DNA, which is inserted at a new site in the genome. It does not have an infective (viral) form. -they contain long terminal repeats (LTRs) are referred to as retrotransposons, while non-LTR-containing retrotransposons are referred to as retroposons. |
| What is an insertion sequence (IS) ? | -is a transposon that codes for the enzyme(s) needed for transposition flanked by short inverted terminal repeats. -The target site at which an insertion sequence is inserted is duplicated during the insertion process to form two repeats in direct orientation at the ends of the transposon (direct repeats). |
| What's a transposase? | -The enzyme activity involved in insertion of transposon at a new site. -The length of the direct repeat is 5 to 9 bp and is characteristic for any particular insertion sequence. |
| How does Transposition Occur? | Most transposons use a common mechanism in which staggered nicks are made in target DNA, the transposon is joined to the protruding ends, and the gaps are filled |