Klausurfragen Genetik/Molekularbiologie

• Class I TE: self-replicating transposons

• Class II TE: self-excision and migration transposon

• Genetic polymorphism is co-existence multiple alleles at a locus (across individuals)

• Repetitive DNA sequences can be divided into two classes and respective subclasses:

  • Tandem repeats (within a genomic region): Satellites, Minisatellites, Microsatellites

  • Dispersed Rrepeats (across the whole genome): Retrotransposons, DNA transposons

• Moderately repetitive dna: 10-100 times repetition of relatively short sequences; dispersed throughout the genome; they have high percentage of transposons

• Highly repetitive dna: very short sequences, typically shorter than 100bp, present many thousand of times

• Homologous genes are genes derived from a common gene ancestor

• Orthologous genes: Genes that are duplicated in different species through speciation events; share a common ancestral gene and may have similar functions in different organisms

• Paralogous genes: Genes that are duplicated within the same species and share a common ancestor

  • In-paralogous genes: Genes that result from a gene duplication event within the same species lineage; often found in tandem arrays on a chromosome and may have diverged in function over time

  • Out-paralogous genes: Genes that result from a gene duplication event followed by speciation (Artbildung), leading to the presence of paralogous genes in different species

• Nucleosome = basic structural subunit of chromatin

• Complex of DNA + proteins that make up chromosomes in eukaryotic cells (“bead-like" structure) that helps to compact and organize the long strands of DNA into a more condensed structure

• Role in regulating gene expression by controlling access to the DNA for transcription factors and other regulatory proteins

• A single nucleosome consists of about 200bp of DNA sequence wrapped around a core of histone proteins, including two copies each of histones H2A, H2B, H3, and H4 (octameric structure) and a linker H1. H1 is a linker between individual nuclear core particles.

• Histone modifications: Chemical modifications such as acetylation, methylation, phosphorylation, and ubiquitination of histone proteins can alter the structure of chromatin and affect gene expression

• Chromatin remodeling complexes: These protein complexes can slide, eject, or restructure nucleosomes to allow access to DNA for transcription factors and other regulatory proteins

• DNA methylation: Addition of methyl groups to DNA can influence chromatin structure by recruiting proteins that either promote or inhibit gene expression

• Amphipathic alpha helix

• leucine in every 7th position

• Is a structural feature in DNA-binding proteins that consists of repeating leucine residues every seventh position along an alpha-helix

• Allows protein dimerization via interactions between hydrophobic surfaces of two leucine zipper proteins

• Adjacent positively charged basic region makes DNA contact (bZIP)

• 2 form a stable protein complex that can bind specific DNA sequences

• Heterochromatin: more densely packaged, located at centromere and pericentromere regions; transcriptionally inactive (dna closed/silent)

• Euchromatin: less densely packaged, located along the length of chromosome arms; transcriptionally active (dna open/ active)

• Epigenetic silencing (DNA methylation, histone modifications, siRNA production)

• Danger: Transposons can cause inactive or unstable alleles, mutations or triggered immune response without a pathogen being present

• The nucleosome is the basic structural subunit of chromatin, consisting of about 200 bp of DNA and an octamer of histone proteins.

• Nucleosome core particle = 4 diff. Histones present as dimers (H2A, H2B, H3, H4) with 2 winds of DNA (= 166 Bp DNA) around the histones

• H1 monomer is associated with linker DNA (34 Bp)

• H1 is a linker between individual nuclear core particles

• Nucleosome = 200Bp DNA with all 5 diff. Histones (H1-H4)

• Definition = Any potentially stable and heritable change in gene expression that occurs without changes in DNA sequence.

• Phenomenon: Genomic imprinting, DNA methylation, Histone modification

• Nucleosome remodeling

• Utilization of different histone variants

• DNA methylation, (predominantly N-terminal) histone modification (methylation, acetylation, phosphorylation, ubiquitylation, sumoylation, ADPribosylation, …)  

• PTMs can affect the chromatin directly by a structural change or indirectly by inhibit the binding of a negative-acting factor/recruit a positive-acting factor

• The Shine-Dalgarno sequence can be found in prokaryotes only.

• The Shine-Dalgarno sequence is a polypurine sequence AGGAGG which is centered about 10 bp before the AUG initiation codon on bacterial mRNA.

• It is complementary to the sequence at the 3′ end of 16S rRNA.

• Base pairing between the SD sequence and rRNA is required for proper positioning of the ribosome thus for translation initiation.

• Replication converts a fully methylated site to a hemimethylated site

• Only the parental strand is methylated

• DNA methyltransferase = Enzyme that adds a methyl group to a specific target sequence in DNA. (in mammals: Dnmt1)

• The state of methylated sites could be perpetuated by an enzyme (Dnmt1) that recognizes only hemimethylated sites as substrates

• DNA methylation in the CG context is typically inherited across cell divisions through maintenance methylation “maintenance methyltransferase”

• Complex: siRNA associate with ARGONAUTE effector complex, forming the ribosome-inactivating silencing complex (RISC) complex

• Mediator: mRNA annealing with miRNA is mediated by ARGONAUTE

• Alternative effects: mRNA degradation, inhibition of translation, DNA methylation of complementary strand

• = DNA-protein complexes or specialized chromatin structures that typically contain hypersensitive sites

• Insulator elements are able to block passage of any activating or inactivating effects from enhancers, silencers, and other control elements

• Insulators define transcriptionally independent domains by providing barriers against the spread of heterochromatin and

• Their effectiveness can be influenced by the direction in which they are oriented

• A-Site: Entry of aminoacyl-tRNA

• P-Site: Binds peptidyl-tRNA

• E-site: Exit of deacetylated tRNA

• The regulation of translational activity in mRNAs is crucial for controlling protein synthesis in response to varying cellular conditions.

• It allows cells to finely tune the production of specific proteins, enabling them to adapt to environmental changes, developmental cues, and various physiological states

• Temporal Control of Developmental Processes

• Energy Conservation

• TFs and TRs play roles in regulating transcription, TFs specifically refer to proteins that bind DNA and directly modulate gene expression, whereas TRs encompass a broader range of proteins involved in transcriptional regulation through various mechanisms beyond direct DNA binding.

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• Initiation, Elongation, Termination

• Multiple codons encode same AA often differ 3rd base position

• Pairing between the 1st base of the anticodon and the 3rd base of the codon can vary from standard Watson-Crick base pairing according to wobble (e.g., G-U pairing)

• Chromatin-Immunoprecipitation (ChIP) with antibody that recognizes specific modification followed by analysis of precipitated DNA

• Quantitative PCR

• Microarray hybridization (ChIP-chip)

• Next generation sequencing (ChIP-seq)

• DNAseI hypersensitivity

• Mnase hypersensitivity

• ChIP with histone antibodies

• Chromatin remodelers remodel the chromatin structure by displace or reorganize nucleosomes

• The process is energy dependent, the source of energy is the ATP hydrolysis

• Remodeling occurs in conjunction with activation of genes for transcription

• RNA interference = RNAi = Silencing of gene expression due to the action of short RNA molecules (21-24 nt).

• There are different forms of RNA interference, meaning silencing can happen in various forms.

• Binding of the respective RISC complex to the mRNA can lead to: mRNA cleavage, DNA methylation, Translation inhibition

• How does RNAi work:

  1. Required: (long) double- stranded RNA molecule (dsRNA)

  2. dsRNA is processed by DICER into short RNA (siRNA, 20-25nt)

  3. siRNA associate with ARGONAUTE effector complex (RISC)

• 4. Complementary mRNA (sequence specificity!) is bound by RISC and inhibited

•  Transgene (hairpin constructs)

•  Complex integration of transposons

•  Natural cis-Antisense Transcripts

•  RNA-dependent RNA Polymerase (RDR)

A method to identify such SNPs is called "genome-wide association study" = GWAS, In GWAS, the entire genomes of e.g. healthy people and patients are scanned for SNPs, and SNPs associated with the disease are identified 

• Inbreeding increases homozygosity, leading to a higher frequency of identical alleles and reduced genetic diversity within a population.

• This can elevate the expression of recessive alleles, increase the risk of genetic disorders, and result in the fixation of alleles.

• Overall, inbreeding has negative consequences on population health and adaptability

• Relationship between a given genotype and its phenotypes in different environments.

• It describes the range of phenotypes that a single genotype can give rise to under diff. environments

• It is known for zero human traits due to complexity and ethical reasons.

• RNA polymerase binding in bacteria involves the sigma factor recognizing the core promoter region containing the −35 region and the −10 (pribnow) region.

• Only possible with high-density genotyping

• Only works, if the causal polymorphism is at a reasonable frequency in the population (>5%)

•  Does not work under allelic heterogeneity

• Individual loci identified often only contribute little to phenotype or probability of disease

• Recombinant inbred lines (RILs) are a type of genetically stable and reproducible population that is often used in quantitative trait locus (QTL) mapping.

• Homogeneous genetic background:

  • RILs are derived from the same two parental lines that have been intercrossed and subsequently selfed for many generations, resulting in a genetically homogeneous population. This genetic uniformity reduces the influence of background genetic variation, environmental factors, and epistasis on trait expression, making it easier to detect the effect of QTLs.

• Permanence:

  • RILs are permanently fixed and can be maintained as a stable population, which allows for replication and comparison of QTL mapping studies across time and different experimental conditions. This is particularly useful for studies that require large-scale phenotyping and genotyping across multiple environments, as it ensures that the genetic variation and environmental conditions are consistent across experimental replicates.

they are likely to be subject to the forces of genetic drift and natural selection, which can influence their fate

= Narrow-sense heritability

• is the proportion of the overall phenotypic variance that is due to additive genetic variance

• genetic variance $s_g^2$ is composed of additive genetic variance ($s_a^2$) and dominance-dependent variance ($s_d^2$)

• p = g + e

• only additive genetic variance is relevant for selection and breeding

the next step for fine mapping a gene involves narrowing down the region containing the gene of interest:

→ Fine Mapping with Recombinant Inbred Lines (RILs)

• repeated measurements → more accurate phenotype estimate for a given genotype

• allows studying genotype and environment interactions

• only have to be genotyped once → for additional traits, only phenotyping required

1. DS-break

2. 3’-ends are processed to overhang

3. repair synthesis/ strand invasion

4. d-loop formation → second end capture, synthesis and ligation

5. HJ-resolution

   → DSB repair with crossing over

   → DSB repair without crossing over

• homologous recombination:

  • sequence is over whole sequence homologous/ identical

• specialized recombination:

  • sequence is not/ not over entire sequence homologous

  • requires recombinase

  • has special att flaring sides that allow recombination → gateway cloning

gateway cloning

Si32

assembly of specific vectors

→ gene conversion.

• a molecular process in which one allele of a gene is converted into another allele, resulting in non-Mendelian inheritance patterns

• It is based on the exchange of genetic information between homologous chromosomes, resulting in the conversion of one allele into another

• clamp loader places the processivity subunits on DNA, where they form a circular clamp around the nucleic acid

• at least one catalytic core is associated with each template strand

→ the clamp loader controls association of the core enzyme with the DNA

• processivity = the ability of an enzyme to perform multiple catalytic cycles with a single template instead of dissociating after each cycle

initiator of replication

→ present in nucleus prior to replication and removed/ inactivated during replication

mostly in eukaryotic cells

→ ensures that only one replication is started per cycle!

Licensing factor

• DNA polymerases often have a 3′–5′ exonuclease activity that is used to excise incorrectly paired bases.

• The fidelity of replication is improved by proofreading by a factor of about 100.