Molecular Biology: Genetics, Transcription, Translation, and Recombinant Technologies

Comprehensive vocabulary flashcards covering the molecular biology of genomes, DNA replication, DNA repair, PCR, DNA sequencing, molecular cloning, transcription, RNA processing, RNA interference, translation, epigenetics, and protein analysis methods.

Reference Genome

A representative digital nucleic acid sequence database of a species' genome, assembled from one or a small group of individual donors, against which individual genomes can be aligned and compared.

Pangenome

The entire gene set and sequence variation present across all individuals within a given species.

Nucleosome

The fundamental subunit of eukaryotic chromatin, composed of approximately $147\,\text{bp}$ of DNA wrapped around an octamer core of histone proteins (two copies each of H2A, H2B, H3, and H4).

Chromatosome

A chromatin sub-structure consisting of a nucleosome core particle bound and sealed at its entry and exit DNA sites by one molecule of linker histone H1.

Euchromatin

A relatively decondensed, open state of chromatin that contains transcriptionally active genes accessible to RNA polymerase and transcription factors.

Heterochromatin

A highly compacted, dense state of chromatin that is transcriptionally repressed and typically concentrated at centromeres, telomeres, and the nuclear envelope.

Semiconservative Replication

The mechanism of DNA replication, proven by Meselson and Stahl, in which each replicated double helix contains one original parental strand and one newly synthesized daughter strand.

Topoisomerase

An enzyme that relieves torsional strain and overwinding ahead of the replication fork by transiently cleaving and resealing one or both strands of DNA to serve as a swivel.

Okazaki Fragments

Short, discontinuous segments of DNA synthesized on the lagging template strand in the $5' \rightarrow 3'$ direction during replication, initiated by short RNA primers.

Proofreading

The intrinsic error-correction ability of DNA polymerase mediated by its $3' \rightarrow 5'$ exonuclease catalytic domain, which excises mispaired nucleotides at the growing $3'$ end before continuing synthesis.

Base-Excision Repair (BER)

A cellular DNA repair mechanism initiated by a DNA glycosylase that cleaves damaged bases (such as uracil formed by cytosine deamination) to yield an AP site, which is subsequently processed by AP endonuclease, deoxyribosephosphodiesterase, DNA polymerase, and ligase.

Nucleotide-Excision Repair (NER)

A repair pathway that removes bulky lesions, such as UV-induced pyrimidine dimers, by unwinding DNA, cleaving on both sides of the lesion to excise an oligonucleotide, and resynthesizing the strand.

Homologous Recombination

A high-fidelity repair process for DNA double-strand breaks that uses an undamaged sister chromatid or homologous chromosome as a template, driven by nucleases, RecA/RAD51 strand invasion, and Holliday junction resolution.

Ames Test

A screening assay using histidine-auxotrophic Salmonella bacteria to evaluate the mutagenic potential of chemical compounds by measuring reversion to histidine prototrophy on histidine-deficient media.

Polymerase Chain Reaction (PCR)

An in vitro enzymatic technique conceived by Kary Mullis for exponentially amplifying specific DNA segments through repeated cycles of high-temperature denaturation, primer annealing, and extension by a DNA polymerase.

Taq Polymerase

A thermostable DNA polymerase isolated from Thermus aquaticus that resists heat denaturation at $95^\circ\text{C}$, allowing continuous automated thermocycling without requiring enzyme replenishment each cycle.

Annealing Temperature

The temperature phase in a PCR cycle (typically $48^\circ\text{C}-72^\circ\text{C}$) at which short single-stranded oligonucleotide primers hybridize specifically to their complementary sequences on denatured template strands.

Primer Dimer

An artifactual low-molecular-weight PCR byproduct generated when two primers anneal to each other at their $3'$ ends and are extended by DNA polymerase.

Dideoxynucleoside Triphosphate (ddNTP)

A modified nucleotide that lacks a $3'\text{-OH}$ group (as well as a $2'\text{-OH}$), preventing the formation of subsequent phosphodiester bonds and acting as a chain-terminator in Sanger sequencing.

Sanger Sequencing

A classic DNA sequencing method that uses DNA polymerase, a specific primer, dNTPs, and chain-terminating ddNTPs to produce nested fragments that reveal nucleotide order when separated by size.

Next-Generation Sequencing (NGS)

High-throughput massively parallel sequencing technology, such as Illumina, that determines sequences within millions to billions of surface-immobilized clonal clusters using reversible fluorophore-labeled chain-terminating nucleotides.

Electropherogram

A fluorescent trace output from automated capillary Sanger sequencing displaying four-colored peaks that correspond to the nucleotide sequence read in order from smallest to largest fragment.

Restriction Endonuclease

A bacterial enzyme that recognizes a specific palindromic DNA sequence and cleaves the phosphodiester backbone, producing either blunt ends or overhanging sticky ends.

Multiple Cloning Site (MCS)

A synthetic polylinker region in a cloning vector engineered with closely spaced, unique recognition sites for various restriction enzymes to facilitate insertion of foreign DNA.

Blue/White Screening

A recombinant selection technique where foreign DNA insertion into the plasmid lacZ multiple cloning site disrupts $\beta$-galactosidase function, resulting in white recombinant colonies on X-gal plates.

DNA Methylase

A bacterial host enzyme (such as Dam or Dcm in E. coli) that adds methyl groups to specific adenine or cytosine sequences to protect host DNA against restriction endonuclease cleavage.

Recombinant DNA

A DNA molecule formed in vitro by cutting and joining genetic sequences from different biological sources using restriction enzymes and DNA ligase.

RNA Polymerase II

The eukaryotic multisubunit enzyme that transcribes all protein-coding messenger RNA (mRNA) precursors, as well as most small nuclear RNAs (snRNAs) and microRNAs (miRNAs).

Preinitiation Complex (PIC)

The complete assembly formed at eukaryotic core gene promoters that includes TFIID, TFIIB, TFIIF, RNA Polymerase II, TFIIE, TFIIH, and the Mediator complex, necessary to initiate basal transcription.

TATA-Binding Protein (TBP)

A subunit of general transcription factor TFIID that recognizes the TATA box in the promoter minor groove and sharply bends the DNA double helix to promote promoter opening.

TFIIH

A multisubunit general transcription factor containing two ATP-dependent DNA helicases that melt promoter DNA and a kinase subunit that phosphorylates Ser5 of the RNAPII C-terminal domain.

Mediator Complex

A large multisubunit protein coactivator ($> 20$ subunits) that acts as a bridge transmitting regulatory signals from DNA-bound transcription factors to RNA Polymerase II and the preinitiation complex.

Enhancer

A cis-acting regulatory DNA sequence that binds gene-specific activator proteins to stimulate transcription, functioning independently of orientation and across distances up to hundreds of kilobases from the promoter.

5' Cap

A post-transcriptional modification added to the $5'$ end of eukaryotic pre-mRNAs consisting of 7-methylguanosine attached via a $5'-5'$ triphosphate bridge, which protects transcripts from exonucleases and facilitates ribosome recruitment.

Spliceosome

A large ribonucleoprotein machinery comprising five small nuclear ribonucleoproteins (U1, U2, U4, U5, and U6 snRNPs) that recognizes consensus splice sites and excises introns while ligating exons.

Alternative Splicing

A process of generating diverse mature mRNA isoforms from a single pre-mRNA transcript by differential selection of splice sites and inclusion/exclusion of exons, expanding proteomic diversity.

RNAPII C-Terminal Domain (CTD)

The carboxyl-terminal domain of the largest subunit of RNA polymerase II, composed of tandem repeats of the consensus heptapeptide $\text{Y}_1\text{S}_2\text{P}_3\text{T}_4\text{S}_5\text{P}_6\text{S}_7$, whose phosphorylation cycle orchestrates capping, splicing, and polyadenylation.

Dicer

An RNase III-family endonuclease that cleaves double-stranded RNA precursors into short $20-25\,\text{nt}$ double-stranded RNA fragments (siRNAs or miRNAs) featuring two-nucleotide $3'$ overhangs.

RNA-Induced Silencing Complex (RISC)

A ribonucleoprotein effector complex incorporating an Argonaute protein that uses a single-stranded guide siRNA or miRNA to bind complementary target mRNAs, mediating transcript degradation or translational repression.

Co-suppression

The reversible, homology-dependent silencing of both an introduced transgene and a homologous endogenous gene at the post-transcriptional level, discovered by Carolyn Napoli and Richard Jorgensen in chalcone synthase petunia experiments.

Reverse Transcriptase

An RNA-dependent DNA polymerase, identified in retroviruses by David Baltimore and Howard Temin, that synthesizes complementary DNA (cDNA) from an RNA template.

Complementary DNA (cDNA)

A double-stranded DNA copy of a mature mRNA transcript generated enzymatically in vitro using reverse transcriptase and an oligo(dT) primer, containing coding exons without genomic introns.

Quantitative Reverse-Transcription PCR (qRT-PCR)

A real-time method combining reverse transcription with PCR that uses fluorescent detection (e.g., SYBR Green) to quantify mRNA abundance, where lower cycle threshold ($C_t$) values indicate higher initial transcript levels.

Chromatin Immunoprecipitation (ChIP)

An experimental technique used to identify protein-DNA interactions in vivo by chemically crosslinking proteins to DNA, sonicating chromatin, immunoprecipitating with specific antibodies, and analyzing co-purified DNA fragments via qPCR or sequencing (ChIP-Seq).

Electrophoretic Mobility Shift Assay (EMSA)

An in vitro assay used to detect specific binding between a protein and a labeled DNA probe, based on the slower electrophoretic migration of a DNA-protein complex compared to unbound free DNA.

Histone Acetyltransferase (HAT)

An enzyme (e.g., Gcn5p/p55) that transfers acetyl groups from acetyl-CoA to specific lysine residues on histone amino-terminal tails, neutralizing positive charges to decondense chromatin and promote transcription.

Histone Deacetylase (HDAC)

An enzyme that removes acetyl groups from lysine residues on histone tails, restoring positive charges that favor condensed heterochromatin and transcriptional repression.

5-Methylcytosine

A modified base produced by the enzymatic addition of a methyl group to the fifth carbon of a cytosine ring, functioning as a primary repressive epigenetic mark at gene promoters when present in CpG dinucleotides.

Bisulfite Sequencing

A laboratory method used to map cytosine methylation at single-base resolution, wherein sodium bisulfite converts unmethylated cytosines to uracils while methylated cytosines (5-methylcytosines) remain unaffected.

CpG Island

A GC-rich DNA region with a high frequency of cytosine-phosphate-guanine dinucleotides, commonly situated within promoter regions where methylation leads to stable transcriptional silencing.

Degenerate Genetic Code

The characteristic of the standard genetic code where multiple synonymous triplet codons specify the same amino acid, with only methionine (AUG) and tryptophan (UGG) specified by single codons.

Wobble Hypothesis

Francis Crick's proposed mechanism of non-standard base pairing occurring between the third base ($3'$ position) of an mRNA codon and the first base ($5'$ position) of a tRNA anticodon, allowing fewer than 61 tRNAs to read all sense codons.

Open Reading Frame (ORF)

A continuous sequence of nucleotide triplets beginning with an AUG start codon and ending with an in-frame stop codon (UAA, UAG, or UGA) that has the potential to be translated into a complete polypeptide.

Polysome

A structure formed by multiple ribosomes simultaneously translating a single mRNA molecule, typically circularized by interactions between poly(A)-binding protein (PABP) and initiation factors eIF4E/eIF4G.

Peptidyl Transferase

The ribozyme activity located in the large ribosomal subunit (mediated by the $23\text{S}$ rRNA in prokaryotes or $28\text{S}$ rRNA in eukaryotes) that catalyzes peptide bond formation between the P-site peptidyl-tRNA and the incoming A-site aminoacyl-tRNA.

Amino Acid

The organic building block of proteins, consisting of a central alpha carbon bound to an amino group ($\text{-NH}_2$), a carboxyl group ($\text{-COOH}$), a hydrogen atom, and a variable chemical side chain (R group).

Translational Fusion

A recombinant gene construct where a reporter gene (e.g., GFP) is fused in-frame to the open reading frame of a target gene (with the target's original stop codon removed) to determine protein localization and abundance.

Transcriptional Fusion

A recombinant construct where a gene's promoter is fused directly upstream of a reporter gene without including the original gene's open reading frame, used to examine promoter activity and spatial expression patterns.

Western Blotting (Immunoblotting)

An analytical method in which cellular proteins are separated by SDS-PAGE, transferred to a membrane, and probed with specific primary and enzyme- or fluorophore-conjugated secondary antibodies to identify and quantify protein levels.

Co-Immunoprecipitation (Co-IP)

A biochemical technique to identify physical protein-protein interactions by using an antibody against a target bait protein to selectively isolate it along with bound interacting partners from a complex cell lysate.

Molecular Function vs. Biological Function

The distinction between a protein's direct biochemical activity (e.g., RNA synthesis catalyzed by RNA polymerases) and its broader cellular or physiological role (e.g., synthesizing rRNA vs. mRNA vs. tRNA).