Introduction to Genetics

1
Introduction to Genetics as relevant to this
course

• (Ack Roche Genetics CD-ROM, Mishras notes at
  NYU, )

2
Background (1/18)

• Genome, Chromosome, Genes made up of DNAs
• Genetics research (largely over last 100yrs,
  accelerated in last 30 yrs)
• Has led to important advances in medical science.
• Nucleus of a cell contains chromosomes (made
  up of DNA) and proteins.
• DNA (Deoxy Ribo Nucleic acid)
• Is the genetic material that is inherited.
• Contains the information needed by living cells
  to specify their structure, function, activity
  and interaction with other cells and environment.
• A DNA molecule can be thought of as a very long
  sequence of nucleotides or bases.

3
DNA structure (2/18)

• The Nobel Prize in Physiology or Medicine 1962 --
  Crick, Watson and Wilkins
• for their discoveries concerning the molecular
  structure of nucleic acids and its significance
  for information transfer in living material
• Made up of 4 different building blocks (so called
  nucleotide bases), each an almost planar
  nitrogenic organic compound
• Adenine (A)
• Thymine (T)
• Guanine (G)
• Cytosine (C)
• Base pairs (A -- T, C -- G)

4
DNA Structure cont. (3/18)

• Base pairs (A -- T,C -- G) are attached to a
  sugar phosphate backbone to form one of 2
  strands of a DNA molecule.
• Phosphate ((PO4) -3)
• Deoxyribose
• Two strands are bonded together by the base pairs
  (A T, C G).
• Results in mirror image or complementary strands,
  each is twisted (or helical), and when bonded
  they form a double helix.
• Direction of each strand (5 meaning beginning or
  3 meaning end of the strand)
• 5 and 3 refer to position of bases in relation
  to the sugar molecule in the DNA backbone.
• Are important reference points to navigate the
  genome.
• 2 complementary strands are oriented in opposite
  direction to each other.

5
DNA Structure
6
Genome Size
Species Genome Size (in base pairs) No. of Chromosomes
E. Coli 4.64 X 106 1
S. Cerevisae (yeast) 1.205X107 16
C. Elegans (nematode) 108 11/12
D. Melanogaster 1.7X108 4
M. Musculus 3 X 109 20
H. Sapiens 3 X 109 6 feet when completely stretched out 23
A. Cepa (onion) 1.5X1010 8
7
DNA hybridization (DL 3/18)

• Hybridization between complementary DNA sequences
  to form a double stranded DNA molecule.
• One of the most important DNA technology
• Applications of Hybridization
• PCR (Polymerase Chain Reaction)
• Enzymetically generating millions of copies of a
  tiny amount of a particular nucleic acid
  sequence.
• Northern blots analysis
• Possible to study (in a semi-qualitative manner)
  the level of transcription of a particular gene.
• DNA Microarrays
• Can interrogate the level of transcription of
  several thousand of different genes in one sample
  in one experiment.

8
PCR (Polymerase Chain Reaction) (DL 3/18)

• PCR allows selected amplification of a DNA
  sequence.
• Only a tiny amount of DNA is necessary to obtain
  a PCR product (a drop of blood or less is
  enough).
• Complementary DNA primers need to be designed.
• For this the DNA sequence flanking the target
  sequence needs to be known in advance.
• Primers are short synthetic DNA sequences of
  about 20 bases (so called oligonucleotides) that
  can specifically hybridize to a unique
  complementary DNA sequence.
• The approach
• Genomic DNA (the template), Primers (the
  starters), deoxynucleotides (building blocks), a
  special DNA polymerase that is resistant to heat
  (the motor of the reaction) are mixed together in
  one reaction tube.
• Reaction takes place in a thermocycler (an
  apparatus that allows one to precisely heat and
  cool the reaction).
• DNA is heated to almost boiling temperature which
  separates the 2 strands (whole process is called
  heat denaturation)
• Cooling of the mixture allows the primer to bind
  to their complementary sequence of the genomic
  DNA.
• Once the primers bind the DNA polymerase uses
  them as the start site to generate a copy of each
  strand of the targeted gene fragment building 2
  new double stranded molecules.
• Doing it (denaturation followed by cooling) 30
  times, results in 230 109 (1 billion) copies.

9
Northern Blot analysis

• The complete RNA content from a sample is
  separated according to size by electrophoresis.
• Usually done in a sheet of agarose (similar to
  gelatine)
• In response to electric current, larger molecules
  move slower, and smaller move faster, thus
  separating different RNA molecules by size.
• Then RNAs are transferred from gel to a filter
  membrane (blot)
• Blot is then exposed to a solution containing a
  nucleic acid (probe) complement to the sequence
  whose presence in the blot one wants to
  interrogate.
• The probe may be cRNA or cDNA with detectable
  marking (radioactive isotope or a fluoroscent
  tag)
• If the targeted sequence is present in the blot
  then the probe hybridizes and sticks to the blot
  at the location where the targeted sequence is
  located.
• After washing off of excess probe -- a signal is
  detectable and its specificity can be checked
  based on the expected size of the RNA that will
  correlate with how far it has migrated during
  electrophoresis.
• With this method It is possible to study in a
  semiqualitative manner the level of transcription
  of a particular gene.
• Comparison of the results from different samples
  (e.g. different organs etc.) provides information
  about the transcriptional regulation of the gene.

10
DNA Structure cont. (4/18)

• The order of nucleotide bases along a DNA strand
  is known as the sequence.
• The genetic information is encoded in the precise
  order of the base pairs.
• DL
• GenBank database http//www.ncbi.nlm.nih.gov/Entre
  z/
• Human genome project http//www.genome.gov/page.cf
  m?pageID10001694
• DNA sequencing
• Is the process designed to precisely determine
  the sequence of bases in the DNA.

11
Cells, DNA and genome (5,6,8/18)

• During cell division (Mitosis) the entire DNA of
  the cell is copied
• 2 strands separate, complementary strands are
  generated.
• Two duplicate DNA sequences are produced.
• Genome an organisms total DNA content
• Diploid cells cells that carry 2 genome copies
• Haploid cells have a single copy of the genome
• Reproductive germs cells (gametes), i..e., egg
  sperm cells
• Human genome consists of
• 22 autosomal chromosomes (same in males and
  females)
• 2 sex chromosomes X and Y (males XY, females XX)

12
Structure of Chromosomes (7/18)

• Center is called centromere.
• Two ends called Telomere.
• Center separates two arms
• Short arm p
• Long arm q.

13
Structure of genes 9-11/18

• Genes are those parts of the genome that contain
  the information necessary for the building of
  proteins. (size100-several million base pairs)
• Exon (coding sequence), Intron (non-coding
  sequence), regulatory region (at the two ends
  for regulating how actively protein is to be
  synthesized from them)
• Eukaryotes (organisms whose cell have nucleus)
  have genes segmented into exons and introns
• Introns can occur between individual codons or
  within a single codon.
• Promoter (a regulatory element in the 5 end)
• Consists of several short sequences which are
  consensus binding sites for a number of proteins
  called transcription factors.

14
(DL 10/18)

• Prokaryotes (do not have nucleus) genes are not
  segmented to exons and introns.
• Eukaryotes (normally segemented to exons and
  introns)
• Except mitochondrial genes a few nuclear genes.
  
• During gene expression exons and introns are
  transcribed to form a pre-mRNA
• RNA splicing -- removes introns and exons and
  produces mature mRNA molecule that codes for a
  polypeptide.
• Exons sequences that are represented in the
  mature mRNA
• May or may not code for a protein
• Eg. Exons at the 3 or 5 end of mRNA may not be
  translated to proteins

15
Some Genes (from Mishras slides)
Gene Product Organism Exon Length Introns Intron Length
Adenoshine deaminase Human 1500 11 30,000
Apolipoprotein B Human 14,000 28 29,000
Erythropoietin Human 582 4 1562
Thyroglobulin Human 8500 40 100,000
a-interferon Human 600 0 0
Fibroin Silk Worm 18,000 1 970
Phaseolin French Bean 1263 5 515
16
Some human gene locations (From Mishras slides)
Genes chromosome
Insulin 11
Galactokinase 11
Viral oncogene homologues
C-sis 22
C-mos 8
C-Ha-Ras-1 11
C-myb 6
Interferons
a b luster 9
g 12
Genes chromosome
a-globin cluster 16
b-globin cluster 11
Immunoglobulin
k (light chain) 2
l (light chain) 22
Heavy Chain 14
Pseudogenes 9,32,15,18
Growth Hormone gene cluster 17
Thymidine kinase 17
17
Gene expresion (12/18)

• Gene expression (Transcription and Translation)
  from genes to making proteins the 2 step process
• Transcription genetic information in DNA is
  copied into messenger RNA (mRNA)
• Translation mRNA is used as a template to
  synthesize a protein.

18
Central Dogma

• Due to Francis Crick 1958 states that these
  information flows are all unidirectional
• The central dogma states that once information'
  has passed into protein it cannot get out again.
  The transfer of information from nucleic acid to
  nucleic acid, or from nucleic acid to protein,
  may be possible, but transfer from protein to
  protein, or from protein to nucleic acid is
  impossible. Information means here the precise
  determination of sequence, either of bases in the
  nucleic acid or of amino acid residues in the
  protein.

19
Transcription (13/18)

• RNA (Ribonucleic acid)
• Similar to DNA (except for a chemical
  modification of the sugar backbone)
• Instead of T contains U (Uracil) which binds with
  A.
• Is not double stranded but single stranded
• RNA molecules tend to fold back on themselves to
  make helical twisted and rigid segments.
• RNA is synthesized
• By unwinding the DNA double helix separating the
  2 strands.
• Using one of the strands as a template along
  which to build the RNA molecule
• Accomplished by Enzyme RNA polymerase (binds to
  promoter and copies or transcribes the gene in
  its full length)
• Resulting molecule is called Pre-mRNA
• Single stranded pre-mRNA is then procesed.
• Splicing (mediated by spliceosome consisting of
  RNA and proteins) removes the introns.
• Ends modified (Capping modifies 5 end and
  Polyadenylation adds adenines at the 3 end) to
  enhance stability

20
Translation (14/18)

• mRNA is used as a template to synthesize a
  protein.
• Translation takes place outside the nucleus in
  the cytoplasm within organelles called
  endoplasmic reticulum.
• Except for the 5 3 end of the mRNA (which are
  non-coding) the rest of the molecule codes for 1
  protein
• Proteins made up of aminoacids
• 20 different aminoacids used to build proteins in
  humans
• Each encoded by one or more sets of 3 nucleotides
  (called triplets or codons)
• Initial codon is always AUG (coding for
  methionine)
• Translation is terminated by one of 3 stop
  codons.
• Translation process is carried out by ribosomes
  which scan the mRNA, build the polypetide chain
  from aminoacids supplied by transport RNAs
  (tRNA).
• Starts at a particular location of the mRNA
  called the translator start sequence (usually
  AUG)
• tRNA (transfer RNA) are made up of a group of
  small RNA molecules each with specificity for a
  particular amino acid.
• tRNAs carry the aminoacids to the ribosomes, the
  site of protein synthesis, where they are
  attached to a growing polypetide.
• Translation stops when one of UAA, UAG or UGA is
  encountered

21
Post-translational modification (DL)

• The polypetide chain that results from mRNA
  translation is often subject to chemical
  modifications. Eg.
• Glycosylation, phosphorylation, hydrooxylation
• Addition of lipid groups (eg. Fatty acyl or
  prenyl groups)
• Addition of co-factors (e.g. a heme molecule)
• Or proteolytic cleavage
• The type of modification a protein undergoes
  depends on its function and sub-cellular location.

22
Genetic Code (15/18)

• The combination of nucleotides that build the
  different codons represents the genetic code.
• Codon 3 nucleotides 4 kinds of nucleotides. So
  4X4X4 64 possible codons.
• But 20 amminoacids start stop.
• So several codons can specify the same aminoacid.
  (genetic code is degenerate)
• Start codon (AUG) and Stop codons (UAA, UAG,
  UGA).
• Open reading frame (ORF) the sequence of
  nucleotides between and including the start and
  stop codons.
• The Nobel Prize in Physiology or Medicine 1968
  Holley, Khorana and Nirenberg
• for their interpretation of the genetic code and
  its function in protein synthesis
• http//www.nobel.se/medicine/laureates/1968/

23
Amino Acids with Codes (From Mishras slide)

• A Ala alanine GC(UACG)
• C Cys cysteine UG(UC)
• D Asp aspertic acid GA(UC)
• E Glu glutamic acid GA(GA)
• F Phe phenylanine UU(UC)
• G Gly glycine GG(UACG)
• H His histine CA(UC)
• I Ile isoleucine AU(UAC)
• K Lys lysine AA(AG)
• L Leu leucine (CU)U(AG) CU(UC)
• M Met methionine AUG
• N Asn asparginine AA(UC)
• P Pro proline CC(UACG)
• Q Gln glutamine CA(AG)
• R Arg arginine (AC)G(AG)CG(UC)
• S Ser serine (AGUC)(UC)UC(AG)
• T Thr threonine AC(UACG)
• V Val valine GU(UACG)
• W Trp tryptophan UGG

24
Biological Function of Proteins

• Enzyme catalysis DNA polymerases, lactate
  dehydrogenase, trypsin
• Transport hemoglobin, membrane transporters,
  serum albumin
• Storage ovalbumin, egg-white protein, ferritin
• Motion myosin, actin, tubulin, flagellar
  proteins
• Structural and mechanical support collagen,
  elastin, keratin, viral coat proteins
• Defense antibodies, complement factors, blood
  clotting factors, protease inhibitors
• Signal transduction receptors, ion channels,
  rhodopsin, G proteins, signalling cascade
  proteins
• Control of growth, differentiation and
  metabolism repressor proteins, growth factors,
  cytokines, bone morphogenic proteins, peptide
  hormones, cell adhesion proteins
• Toxins snake venoms, cholera toxin

25
Differential Gene Expression 17/18

• All cells in the body (that contain a nucleus)
  carry the full set of genetic information, but
  only express about 20 of the genes at any
  particular time.
• Gene expression is selective
• Different proteins are expressed in different
  cells according to the function of the cell.
• Gene expression is tightly controlled and
  regulated.
• The differential expression of genes ensures that
  cells develop correctly and can differentiate
  into and function as specialized cell types. For
  eg. Neurons, muscle cell, or fibroblast.

26
cDNA and gene expression (DL)

• Goal Identify all possible genes expressed in
  one tissue or cell line. (Use cDNA libraries)
• cDNA libraries are prepared from mRNA isolated
  from the cells or tissue being studied.
• cDNA are DNA molecules that are complementary to
  the mRNA sequences in a sample.
• cDNA is synthesized by the enzyme reverse
  transcriptase (RT), that uses the mRNA as a
  tenplate.
• RT is a viral enzyme used by viruses whose genome
  is made of RNA, not DNA.
• A cDNA library represents the collection of all
  genes expressed in a particular cell or tissue
  type.
• DNA sequence ? mRNA sequence ? cDNA sequence
  (much smaller as while generating mRNAs the
  introns are eliminated)
• Hence very useful when trying to isolate a
  particular gene to study the protein it codes.

27
NEXT SEC.

28
Gene Cloning 1/11

• First step in identifying genes and their
  function is to isolate it from the rest of genome
  and produce a large quantity of it (called
  cloning a gene).
• Cloning a DNA fragment using bacteria
• DNA fragment is isolated from the entire genome
  using restriction enzyme.
• These enzymes can cut the DNA (in a staggered
  fashion or straight through) at specific sites
  defined by a short sequence.
• Typically they recognize specific DNA sequences
  of 4, 6, or 8 bases
• These enzymes are found in bacterias, where their
  role is to protect the bacteria from foreign DNA
  by digesting them into smaller pieces
• This fragment is inserted into a vector (like a
  mini-chromosome) using DNA ligase and the
  recombinant product is introduced into bacteria
  (this process is called transformation)
• Cloning vectors are DNA fragments that are able
  to replicate within a cell and allow the addition
  of exogenous DNA.
• They are derived from plasmids, viruses, phages
  or chromosomes.
• Vectors are classified according to the type of
  host cell they can replicate in, or the size of
  the exogenous DNA they are able to carry.
• The bacteria now makes new copies with every cell
  division.

29
DNA Sequencing (DL 1/11)

• It is the process designed to precisely determine
  the sequence of bases in the DNA.
• Involves enzymetically copying the DNA in the
  presence of compounds that terminate this copying
  process in a base specific manner, resulting in a
  mixture of DNA copies that differ in size by one
  base.
• Different technologies are used to resolve the
  mixture and detect the different fragments.

30
Cloning issues (2-3/11)

• Clones from genomic DNA contain introns
  (non-coding sequence) and is very large and
  difficult to analyze for function.
• Alternative start from mRNA. Convert to cDNA and
  clone the cDNA.

31
Gene function characterization(4/11)

• To characterize the function of a gene it is
  important to know the sequence and compare it to
  other sequences in the databases. Identify where
  and under what condition it is expressed and what
  function, if known, it has in other organisms.
• Also do gene expression studies.

32
Gene expression studies (5/11)

• Allow you to understand how a gene is regulated
  in a tissue or a cell type.
• Most useful way of studying gene expression is
  by measuring the levels of mRNA produced from a
  particular gene in a particular tissue.
• Application to understand certain biological
  process it is useful to study the differences in
  gene expression which occur during such
  processes. E.g.
• It is of interest to know which genes are induced
  or repressed, say in the liver, after a
  particular drug is taken.
• Or which genes are expressed in a tumor but not
  in the surrounding normal tissue.
• Some techniques for analyzing mRNA level of a
  single gene or to quantify gene expression
• Northern blots
• Quantitative reverse transcriptase PCR
  (QT-RT-PCR)
• DNA microarrays
• Proteomics (analysis of the protein synthesis
  that results from gene expression)

33
DNA microarrays (6/11)

• Consist of thousands of DNA probes corresponding
  to different genes arranged as an array.
• Each probe (sometimes consisting of a short
  sequences of synthetic DNA) is complementary to a
  different mRNA (or cDNA)
• mRNA isolated from a tissue or cell type is
  converted to fluoroscently labeled mRNA or cDNA
  and is used to hybridize the array.
• All expressed genes in the sample will bind to
  one probe of the array and generate a fluoroscent
  signal.
• A DNA microarray can interrogate the level of
  transcription of several thousand of different
  genes from one sample in one experiment. (One DNA
  microarray experiment reveals the mRNA levels of
  1000s of genes from one tissue or cell type at
  one time point)
• Particularly useful when studying the effect of
  environmental factors on gene expression.
• A fingernail size chip can interrogate 10,000
  different transcripts. Chip has 30-40 different
  probes half of them are designed to perfectly
  match 20 nucleotide stretches of the gene and the
  other half contains a mismatch as a control to
  test for specificity of the hybridization signal.

34
Pharmacogenomics 7/11

• It refers to the study of differential gene
  expression applied to drug discovery and
  optimization.
• Applications (Differential gene expression
  studies in special tissues or cell types may)
• Find new disease mechanisms of a drug
• Discover new drug targets
• Confirm expected action of mechanism of a drug
• Choose from best candidate compound based on
  optimal expression profile.
• Figure out apriori with who will benefit from a
  drug and who wont.

35
Model organisms 9/11

• Indispensable tool to study the function of a
  gene.
• Range from bacteria and yeast to animals amenable
  to genetic modification.
• Worms, insect cells, frog eggs, flies, zebra
  fish, mice, mammalian (human) cell lines.
• In general, more complex the organism more
  difficult to do genetic modification, but more
  relevant the model becomes to humans.