Bacterial IS elements (Insertion Sequence) and equivalent eukaryotic elements have a diagnostic structure: a central protein coding region containing genes for transposition enzymes; flanking Inverted Repeats that serve as recognition sequences for the transposase; and distal flanking short direct repeats. The Inverted Repeats are part of the transposon and are the same for all copies [...]

Mitosis Mitosis is a eukaryotic process whereby a cell divides to produce two daughter cells identical to itself. Mitosis is the nuclear division that results in two daughter nuclei whose genetic material is identical with that of the original nucleus. In multicellular organisms, somatic cells undergo mitosis while germ cells undergo meiosis. Prokaryotic cells lack [...]

Promoters in E. coli were found using bacteriophages like λ and T7, which act very strongly to encode massive quantities of viral proteins. This same idea was expanded to eukaryotes, using adenoviruses (which infect eukaryotic cells). Viral genes express massive amount of proteins and a great model for finding examples of eukaryotic promoter sequences. Nuclear [...]

Initiation RNAP (RNAP) recognizes and binds to promoter region on dsDNA, forming the closed complex. Around the initiation site (+1), the DNA is unwound & becomes single-stranded; the RNAP/ssDNA structure is the open complex. The RNAP transcribes the DNA, but produces about 10 abortive (short, non-productive) transcripts which are unable to leave the RNAP because [...]

Eukaryotes have 3 RNAPs: RNAPI makes rRNA RNAPII makes mRNA RNAPIII makes tRNA & small RNAs Eukaryotes have 5 GTFs Tata Binding Protein (TBP) TFIIH FT TFIID TFIIB TFIIH Eukaryotic promoter has 4 parts B Recognition Element TATA box Initiator Downstream promoter element Initiation TBP recognizes and binds to the tatabox (-31 to -26). TBP [...]

E. coli RNA Polymerase is α2ββ’ω there are three eukaryotic RNA Polymerases. Binding of the RNA polymerase to the gene is highly regulated and requires lots of protein factors even for genes active in all cells. Transcription in eukaryotes requires the assembly of the RNA Polymerase into a very large initiation complex at the gene [...]

What problem does the enzyme telomerase overcome? DNA replication requires an RNA primer to initiate synthesis, which is degraded after priming. The loss of these primers on the lagging strand of the chromosome ends will result in a loss of information with each round of replication. Telomerase is a special enzyme that uses its own [...]

Eukaryotic chromosomes are replicated from multiple origins. Initiation of replication from these origins occurs throughout S phase. Some origins fire in early S, some in late S phase. However, no eukaryotic origin initiates more than once per S phase. S phase continues until replication from multiple origins along the length of each chromosome results in [...]

BK channels (aka Slo channels) are tuned via alternative splicing of α subunit exons, thereby controlling regulatory properties, conductance and voltage sensitivity of the channel. BK Channels are present in muscle tissue and in the cochlea

The nuclear envelope consists of a lipid bilayer. Mitotic Breakdown & Reconstruction Long and fibrous lamin proteins form a layer of structural support for the nuclear envelope. Lamin is phosphorylated in prometaphase, causing a conformational change and the loss of laminal structural properties. Without laminal support, the nuclear membrane breaks apart and absorbs into the [...]

Polyubiquitination marks eukaryotic proteins for degradation by proteasomes. Three enzymes are required for the Ubiquitin to work: E1, the Ubiquitin-activating enzyme; E2, the Ubiquitin-conjugating enzyme; and E3, the Ubiquitin ligase. SCF and APC/C are ubiquitin protein ligase complexes that that control three major transitions in the cell cycle: onset of S-phase through degradation of Sic1 [...]

Human Yeast Activates Overview CAK Kinase Cyclin-CDKs CAK positive phosphorylates CDK1, CDK2 and CDK4. CAK is itself a member of the CDK family and is composed of CDK7, cyclin H and an assembly protein Mat1. Checkpoint controls, inactivate Cdc25C and Cdc25a phosphatases to induce cell-cycle arrest. (link) Cdc25 phosphatase Cyclin-CDKs Involved in activating MPF. Cdc25a [...]

Human Yeast Inhibits Overview Wee1 Kinase Cyclin-CDKs Inhibitory phosphorylation of CDK1 and CDK2. (link INK4 Mid-G1 CDKs INK4s include cyclin-dependent kinase 4 and 6, which have important tumor suppression activity by inhibiting the mid G1 CDKs, thus inhibiting passage through G1. Both genes encoding INK4a are mutated in many human tumors, lessening their ability to [...]

Checkpoint Overview DNA Damage A Midway through G1, ATM/R activates p53, which activates p21CIP, which blocks Mid-G1-Cyclin+CDK (Cyclin-D+CDK4 & CDK6) if DNA damage is detected. DNA Damage B At the start of S-phase, ATM/R activates: p53, which activates p21CIP, which blocks the late G1 cyclin (Cyclin E) and the S-Phase cyclin (Cyclin A) if DNA [...]

How is the dismantling of the nuclear lamina during prophase and its reassembly during telophase accomplished? Where does the nuclear envelope go during mitosis? Long and fibrous lamin proteins form a layer of structural support for the nuclear envelope. Lamin is phosphorylated in prometaphase, causing a conformational change and the loss of laminal structural properties. [...]

Metaphase Checkpoint How does the metaphase checkpoint prevent sister chromatid separation at the onset of anaphase until every kinetochore has become associated with spindle microtubules? In the spindle assembly checkpoint (aka metaphase checkpoint), mitotic arrest deficient 2 (aka Mad2) blocks metaphase until every single kinetochore has properly attached to spindle microtubules. Mad2 exists in an [...]

Human disease mutations that create nonsense mutations do not always produce a truncated protein. Often they lead to rapid mRNA degradation via Nonsense-Mediated Decay. During splicing in the nucleus, the Exon Junction Complex, containing 4 proteins, is deposited ~20 nt upstream from each exon/exon junction in the final mRNA. This marks the junctions and stays [...]

Cycles in M-Phase Promoting Factor (MPF) activity control mitosis. As a protein kinase, MPF likely acts via phosphorylation of the major histone protein H1 and the major nuclear envelope protein lamin. This leads to the degradation of the nuclear envelope and condensation of chromatin into chromosomes in anticipation of mitosis. Found in all organisms, MPF [...]

Step Overview Growth 1 In a diploid eukaryotic cell, there are two versions of each chromosome, one from the mother and another from the father. The two corresponding chromosomes are called homologous chromosomes. Homologous chromosomes need not be genetically identical. During growth 1 (G1), an interphase, phase is the normal growth phase. Chromosomes are highly [...]

Microtubule organizing centers (aka centrosomes) are composed of asters at each end, with centrosomes spanning between them. The tubules that connect to the chromosome kinetochore are called kinetochore microtubules, while the tubules which interact with each other are polar microtubules (aka non-kinetochore microtubules). Microtubules are composed of α- and β-tubulin monomers polymerized to form hollow [...]

An obvious advantage of proteolysis for controlling passage through these critical points in the cell cycle is that protein degradation is an irreversible process, ensuring that cells proceed irreversibly in one direction through the cycle. Step Initiation Overview Early G1 DNA prepreplication complexes assemble at origins. However, they are not activated. Mitotic cyclin-CDKs activate early [...]

Factor Overview E2F E2F is a transcription factor that by itself activates transcription. Rb Rb binds to E2F and represses its activation function. Rb is deactivated upon phosphorylation by mid G1 cyclin-CDKs (and, eventually, late G1 cyclin-CDKs). Cohesin Cohesin is a heterotrimeric complex of Smc1, Smc3 and Kleisin (Scc1). Smc1 and Smc3 form a circle [...]

Mitotic Events Process Genomic Cellular Prophase The genome condenses. Visible chromosomes form. Each chromosome has 2 sister chromatids bound at the centromere by cohesin. Spindle fibers (aka spindle poles) emanate from the centromere. The two centrosomes form G2 sprout microtubules by polymerizing free-floating tubulin. The microtubules repel each other, pushing the centrosomes to opposite ends [...]

Mutations often render a protein unstable, meaning that it can function at lower temperatures but will quickly fall apart at higher temperatures. This feature can be manipulated to identify genes important in different processes; at a lower temperature, the mutant will be wild-type; at a higher temperature, the mutant will reveal its phenotype. Experiments that [...]

Sex-lethal is a sequence-specific RNA binding protein that recognizes a specific UGUUUUUUU element in its target RNAs. It has a Β1,2,3 & 4 domains as well as RNA recognition motifs RRM1 and RRM2. The presence or absence of Sxl in an early embryo will determine whether it develops as a male or a female. Early [...]

Cochlear Hair Cells are tuned to respond to different sound frequencies. These cells are arrayed in a tonotopic gradient, with low frequency responders at the apical end of the cochlea and high frequency responders at the basal end. Birds and reptiles uses alternative splicing of BK Channels as one facet of tuning these hair cells [...]

Humans 3×109 base pairs ~25,000 genes Fruit fly 1.2×108 base pairs ~13,600 genes Round Worm 9.7×107 base pairs ~19,100 genes Bakers Yeast 1.2×107 base pairs ~6000 genes Does gene number correlate with complexity? As shown to the left, it obviously does not. Rather, alternative splicing correlates with complexity. 95% of Human genes are known to [...]

Spinal Muscular Atrophy is the most common genetic cause of infant death, occurring in 1 in 6,000-10,000 births. This is an autosomal recessive disorder caused by mutations in the SMN1 gene, encoding a protein involved in snRNP maturation. Humans have undergone an SMN gene duplication generating the SMN2 gene. Even though it encodes an identical [...]

Nearly all disease genes have mutant alleles which affect splicing of the mRNA, rather than protein coding. Exon skipping can arise via mutations in the 5′ splice site, the 3′ splice site, and/or exonic or intronic splicing enhancers. Activation of cryptic splice sites (thus shortening the exon) can arise via mutated exonic splicing enhancers. Examples [...]

Some mitochondrial, protist and archaeal genes have autocatalytic self-splicing introns (as opposed to nuclear spliceosome-dependent introns). These introns fold into specific secondary and tertiary structures that catalyze their own excision, without proteins. There are two families of these introns — Group I and and Group II — and Group II autocatalytic introns are thought to [...]

Most metazoan genes have multiple exons that must be carefully excised (from the pre-mRNA) and then ligated (to form the mRNA). This RNA splicing occurs in the nucleus, and upon its completion the mRNA is exported to the cytoplasm for translation. Exon definition complexes and spliceosomes begin to assemble during transcription. Some of these complexes [...]

Intron Definition Introns have sequences that directs the splicing apparatus during RNA splicing, part of RNA processing: Introns begin and end with splice sites that conform to consensus sequences. Introns always begin with a GU encompassed within a larger 5’ splice site consensus. Introns always end with the branch point sequence, several pyrimidines and an [...]

What Is the Spliceosome? The spliceosome is a hugely massive complex that catalyzes excision of introns and ligation of exons. In addition to many non-snRNP subunits, the spliceosome consists of 5 small nuclear ribonucleoproteins (snRNPs) that assemble stepwise onto each intron. The snRNPs are small and are composed of: a small nuclear RNA (snRNA) with [...]

The pre-mRNA binds many proteins during transcription. Although collectively called hnRNP proteins, they in fact belong to many different structural and functional families. Like transcription factors, RNA binding proteins can be modular and contain and RNA-binding domain and a protein-protein interacting domain. RNA-Binding Domain RNA-binding domains often recognize short sequences (like a base triplet) and [...]

Class Length Copy # Genome Overview Tandem Repeats Variable 20 – 300 0.3% Encode rRNAs, tRNAs, snRNAs and histones. In some cases, the multiple copies allow increased production of identical gene products – rRNA and histones. In these cases, the genes usually exist as tandem arrays. Allows production of millions of copies of the gene [...]

The most common type of repetitive DNA are interspersed repeats or moderately repeated sequences. These are present as a single copy at very many different loci and can move or jump to new locations.Interspersed repeats account for almost half of human DNA. These do not occur in tandem arrays. Individual copies of the same, or [...]

Class Length Copy # Genome Overview Solitary Genes Variable 1 ~15% Gene Families Variable 2 – ~1,000 ~15% A significant percentage of human genes are members of gene families. In some cases, the multiple copies allow increased production of identical gene products – rRNA. In other cases, the different family members have different but related [...]

DNA fingerprinting (aka DNA profiling) is useful for: paternity testing; establishing twin zygosity; determining bone marrow transplant engraftment; identifying mislabeled pathology species (discomfortingly frequent); pedigree analysis of animals and animal products; and establishing identity of criminals. CODIX is an FBI-administered DNA index that catalogs the DNA fingerprints of individuals. DNA profiling can be done by [...]

Eukaryotes encode long precursor RNA transcripts, which are spliced, polyadenylated and capped to produce a messenger RNA that can be translated. Capping Nucleus Guanosine is added to the 5′ end of the mRNA for stability, transport and translation initiation. Polyadenylation Nucleus The 3’ end of the RNA is cleaved and a Poly-A tail is added [...]

These mutations help determine the transcriptional control region of a gene. The 5′ region keeps getting clipped away until transcription control is disrupted, thus determining the region.

cDNA is single-stranded DNA that has been reverse-transcribed in vitro from cellular mRNA. By soaking a cDNA microarray with labeled RNAs, it is possible to determine which genes are active in a particular cell. A cDNA microarray is a grid of dots, with each dot containing a different cDNA sequence. In the figure below, darker [...]

The in vitro nuclear run-on experiment is a great way for isolated specific RNA transcript. Also, as shown in the last step, the in vitro nuclear run-on experiment can measure transcription of a gene relative to overall cellular transcription. Together with ion exchange chromatography, which showed that almost all RNA transcripts are translated into protein, [...]

Exon shuffling can lead to a common domain being found in a variety of proteins. This shuffling around of mobile elements is thought to have had a profound influence on the evolution of genomes of multicellular organisms. Via Non-Viral Retrotransposon With non-viral retrotransposons, if a LINE has a weak poly(A) signal, then sometimes transcription will [...]

During meiosis, homologous chromosomes are paired and aligned. This allows homologous recombination or crossing over to occur between the maternal and paternal derived chromosomes. Recombination or crossing over of these paired chromosomes increases genetic diversity in the offspring by associating maternal alleles with paternal on the same chromosome. Upon the division of meiosis I, recombinant [...]

There are numerous diseases which arise from expansions in (CAG)n repeats, which encode glutamine. In a coding region, a (CAG)n repeat expansion can either inactivate a protein or exhibit a dominant phenotype due to poly-glutamine’s toxic effects; examples include Huntington Disease and Spinocerebellar Ataxia (SCA) type I. In a non-coding region, a (CAG)n repeat expansion [...]

The 24 types of human chromosome can be distinguished by different staining procedures. Each chromosome has a unique banding pattern, a distinctive pattern of dark bands (stained regions) and light bands (unstained regions). Banding of condensed metaphase chromosomes reveals about 450 different bands. Based on the banding pattern and the location of the centromere, chromosomes [...]

Eukaryotic cells are defined as having a nucleus, thus distinguishing eukaryotes from prokaryotes. The nucleus is an organelle containing the genome and complex machinery to control gene expression, and is separated from the cytoplasm by a double-membrane. From the nucleus, genetic information flows to the cytoplasm for utilization. In contrast, while eukaryotes encase their genome [...]

Cyclin-dependent kinases regulate two important transitions in the eukaryotic cell cycle: entry into S-phase and entry into mitosis. CDKs are controlled at every level: presence (controlled degradation); activity (inhibitors); and even specificity (cyclins). Examples are shown below: Cyclins The specificity of cyclin-dependent kinases is regulated by their cyclins. For example, S. cerevisiae uses the same [...]

Some specialized eukaryotic cells increase cell volume via endomitosis, where DNA synthesis is repeated without cell division and a normal chromosome develops into a giant polytene chromosome results (first observed in Drosophila melanogaster larval salivary glands). Centromeres and telomeres endoreplicate poorly, leading to a bundle of duplicate chromatids (as many as 1,000) stuck at the [...]

A mammalian chromosome is massive, with tens to hundreds of megabases of DNA. The entire haploid human genome contains about 3 billion base pairs of DNA. Only about 5% of this encodes functional RNAs or Proteins or controls their production. Eukaryotic chromosomes are bound to structural proteins to form chromatin. During metaphase, chromatin is highly [...]

Gene expression in prokaryotes and eukaryotes is tightly controlled, mostly via transcriptional control whereby the initiation of transcription is highly regulated. In prokaryotes, the promoter is where repressors and activators bind and it is usually near the gene it regulates. In eukaryotes, the promoter is relatively far away and is bound by transcription factors (the [...]

Sir2 is a histone deacetylase found in all eukaryotes. Sir3 and Sir4 associate with Rap1 and with deacetylated histone tails and with each other. Sir2 associates with Sir4, deacetylating the tails of neighboring histones and thus causing this repressing chromatin structure to “spread” along the chromosomes from the telomere. Below is a biochemical method for [...]

0.34 nm = Distance between base pairs in B-form DNA. 3 · 109 bp = Human Haploid Genome. ~1 m = Length of Haploid Genome. 3-10 · 10-6 m = Length of nucleus. ~2 m = DNA per nucleus. To pack DNA into the tiny nucleus (the DNA packing problem), DNA is tightly wound around [...]

Patients with two defective retinoblastoma (Rb) alleles invariably develop retinoblatomas, leading to its discovery as a tumor suppressor. Why do inactivating mutations in the RB (retinoblastoma) gene contribute to the generation of cancer cells? Rb binds to E2F and represses its activation function. Rb is deactivated upon phosphorylation by mid G1 cyclin-CDKs (and, eventually, late [...]

Fertilization is the union of a haploid sperm and haploid egg to form a single diploid zygote. This cell will develop into an embryo. Unfertilized Egg The egg is quiescent — it performs no DNA synthesis, cell division nor RNA transcription. There is a reduced rate of translation until fertilization. The egg stores a multitude [...]

Mitosis is the division of a parent cell into two daughter cells that are identical to itself. A cell does this to increase population size and to increase genetic diversity. The parent cell duplicates each of its chromosomes before mitosis; these duplicate chromosomes are called sister chromatids, and are attached at their centromere. In animals [...]

A gene is the nucleic acid sequence needed to synthesize a particular gene product. A gene includes more than just the coding region that encodes an RNA transcript; there are also control regions controlling synthesis, processing and translation of the RNA transcript. In prokaryotes, the entire coding region encodes a continuous polypeptide sequence. In eukaryotes, [...]

Transposable elements were first discovered by Barbara McClintock in kernels of corn, where certain mutations caused loss and reinstatement of purple pigment (due to gain and loss of an insertion element that activated pigment genes). The human genome contains ~300,000 DNA transposons, which have extensively accelerated evolution due to the modularity of exons and regulatory [...]