1.2 Genome Annotation

What is gene annotation?

The process of determining which sequences do which tasks. Basically, the human genome was attached with meaningful info that helped us understand what our genomes do

What are some key questions of gene annotation?

Location? Amount? Products? and what lies in the genome other than genes?

What is an ORF?

Open Reading Frame, a reading frame (borrowed from polymerases) that is uninterrupted by stop codons

What does a polymerase recognize while building onto DNA?

Start and stop codon

What are the 3 DNA stop codons?

TAA, TAG, TGA

Which frame is considered an ORF?

Frame 5, it is uninterrupted with no stop codons

How are phylogenetic trees made?

By comparing genomic DNA sequences among organisms

How can you interpret a phylogenetic tree?

• Branch points represent a series of nested common ancestors

• Number at each branch point is millions of years before the present

What is the phylogenetic tree’s purpose?

Helps us to figure out what’s our point of origin in the history of the animal kingdom

What is the importance of conserved genes?

Conserved genes are genes that show very little change across different species, it allows researchers to study gene function in model organisms (mouse, zebrafish, fruit fly) and apply that knowledge to humans.

Why is mRNA not easily sequenced?

• Too rare/unstable (due to U base) to purify

• Sequencing technology is not widely available

How is mRNA made into cDNA?

retroviruses (who have RNA genomes) use reverse transcriptase to copy RNA into cDNA

Why is cDNA more stable than mRNA?

• U → T base

• Single stranded → double stranded

How is mRNA isolated?

Using oligo (dT) probe, which attracts its poly A tails at the 3’ end

What is oligo (dT)?

A probe designed as a single strand consisting of DNA fragments of 20 nucleotides made of dT only

How does reverse transcriptase convert RNA to cDNA?

In vitro synthesis, using oligo(dT) as a primer + AGTC bases

How is the 2nd cDNA strand made from the 1st cDNA?

1. mRNA-cDNA hybrids have mRNA aspect digested with RNase

2. 3’ end of SS cDNA folds back and acts as new primer

3. 1st cDNA is now a template

What is an exome?

The part of the genome corresponding to exons

What is the non-coding parts of DNA?

Introns, centromeres, telomeres, transposable elements

What are gene-rich regions?

Chromosomal regions that have many more genes than expected from average gene diversity over entire genome

Ex: class III region of major histocompatibility complex

What are gene deserts?

Regions that have no identifiable genes

Ex: 5..1 Mb on chromosome 5 with no identified genes

What is the most gene-rich region of the human genome?

Class III MHC complex region contains 60 genes within a 70 kb region

What are protein domains?

Sequence of amino acids that fold into functional units

What are two parts of a protein region?

• Transcription factor, helps and actually performs transcription

• Homeodomain (highly conserved), anchors to polymerase

How is a consensus sequence helpful?

A function of a new protein can be deduced if it contains a domain known to play a role in other proteins

What is exon shuffling?

Over the course of evolution, new genes/proteins are made, creating novel domain architectures due to genomes being under selection pressure

What are domain architectures?

The specific arrangement and combination of protein domains within a protein.

What is a gene family?

Groups of genes closely related in sequence and function

Ex: α-globin (C16) and β-globin (C11) for O2 transport

What is a pseudogene?

Genes that are very similar in their sequence to the actual genes (>99%), but they do not code for proteins or any products

What is a theory on how gene families evolved?

Started with an ancestral globin gene, which then duplicated and diverged down the line, becoming dynamic depending on the evironment/other external factors for survival

• possible explanation for α-globin, β-globin, and pseudogenes

What are orthologous genes?

Arose from the same gene in the common ancestor, usually retain same function

Mnemonic: OSG (ortho, similar genes, not similar functions)

What are paralogous genes?

Arise by duplication, often refers to members of a gene family

Mnemonic: PS (para, same species, not similar functions)

What are homologous genes?

Share a common ancestor, encompasses both orthologous and paralogous genes

What are de novo genes?

Genes without homologs, most likely young genes that evolved recently from intergenic sequences (non-coding.. for now)

What are syntenic blocks?

Colored segments that contain genes whose order is conserved in the mouse genome

What are chromosomal rearrangements?

Where blocks have moved to different places in the mouse genome

How does DNA rearrangement affect the human immune system?

DNA rearrangement of V, D, and J segments results in 1000 different combinations within T-cells, making the immune system vastly diverse

What is an example of DNA rearrangement in the brain?

There are 3 neurexin (protein that binds neuron together at synapse) genes, they have 2 alternative promoters and 5 sites for alternative splicing

• This generates 2000 different mRNAs

What is GenBank?

Database established by the NIH in 1982

• most widely used repository for sequence data

What is RefSeq?

Single, complete, annotated version of a species’ genome

• agreed upon and peer-reviewed for standard comparison

What is BLAST?

A mathematical code that can align to sequences and tell us about similarities and dissimilarities

What is bioinformatics?

Computational tools used to analyze biological data such as DNA and protein sequences. Examples:

• GenBank: public database of sequences

• RefSeq: validated reference sequences

• BLAST: compares sequences

An adult hemoglobin consists of what?

4 polypeptide chains

• 2 alpha globins

• 2 beta globins

What is the purpose of hemoglobin?

Has a heme group (Fe²⁺) reacts with O^2- to carry 4x at a time, as it circulates to all tissues

At what stage during development does the hemoglobin bind more tightly to oxygen?

Embryonic and fetal hemoglobins, so that it can facilitate transfer of O2 from the mother to the embryo/fetus

At what stage during development does the hemoglobin bind less tightly to oxygen?

Adult hemoglobin, to allow delivery of O2 to the organs

What is occurring during the 1st 6 weeks of development on this graph?

ζ and ε are most efficient at binding to O2, thus it is initially elevated for embryo growth

• Since the embryo is still embedded inside maternal tissue, it needs oxygen to survive

What is occurring between 6 weeks of development to just before birth on this graph?

α and γ are rising, β is mostly flat

• Placenta is forming/formed

What is occurring from birth to after on this development graph?

α stays the same level as it was, γ drops immediately, β starts to increase, δ is also present but at very low levels

• Fetus now moved to needing environmental O2 after birth

Why is the order of hemoglobin genes (or any genes) on a chromosome matter?

In this case, it reflects the timing of expression

• Embryonic globin genes are located first.

• Fetal globin genes are next.

• Adult globin genes are last.

What are some aspects of chromosomal organization on a hemoglobin?

• Genes in order

• Genes oriented in same direction

• LCR controls sequential expression of globin genes

What causes hemolytic anemias?

Change in amino acid sequence of alpha- or beta- globin chain, causing destruction of RBC’s

• Ex: sickle cell anemia

What causes thalassemia?

When mutations reduce or eliminate production of one of the 2 globin polypeptides

• Has a range of phenotypes from normal to severe