Unit 1: Chromosomes and Genes4 days

July 31:

Review Topics

• Genome– Human genome = 25,000 genes

• Chromosomes• Karyotype• Locus

(precise position)• Gene map• Cytogenetics

= study of chromosomes and their structure

Clinical Diagnosis• Many disorders can be traced back to

chromosomal anomalies

• This can include the chromosome’s quantity or structure

• Often visible microscopically

• Down syndrome

= trisomy 21

Gene Mapping

• This is one of the main goals of medical genetics today

• Focus on role of specific genes in health and disease

Cancer Cytogenetics

• Genomic and chromosomal changes that lead to the initiation of cancer

• In somatic cells

• Genes may also

determine how

aggressive the

progression of

certain cancers are

Prenatal Diagnosis

• Chromosome and genome analysis is an important procedure for prenatal diagnosis

Human Chromosomes

• Review:– 46 in somatic cells– First 22 = autosomes – Homologous chromosomes or homologues– X vs. Y = sex chromosomes– Different in germline (gametes)– Alleles– Mitochondrial genes

DNA

• Review:– Adenine and Thymine– Guanine and Cytosine– Purines (A&G) vs. Pyramidines (C&T)– Phosphate– Deoxyribose– 50 million to 250 million base pairs per

chromosome– Right handed double helix

Bases

Chromatin

• Most of the time chromatin is distributed throughout the nucleus, and is not especially apparent

• During division, form chromosomes

• Each chromosome is a continuous DNA strand

Histones

• DNA strand wound around histone proteins

• 5 major types of histones

• Form octamers that the DNA helix coils around

• DNA + histone octamer = nucleosome

Mitochondrial Chromosome

• In cytoplasm

• Bacteria-like

• Maternal inheritance

• 37 genes

• Endosymbiont theory

Genes

• Some areas of chromosomes are ‘gene rich’, others are ‘gene poor’

• ~1.5% of base pairs encode for protein production

• Only ~5% is thought to contain regulatory elements

• Only half of the DNA is unique DNA – the other half is repetitive DNA

Repetitive DNA

• Much of the unique DNA’s purpose is unknown

• Typically found in short bursts, a few kilobase pairs or less

• Several categories of repetitive DNA

Karyotypes• G banding

• Karyotypes

Crossing over

August 12:

Proteins and Genes

• Proteome – set of proteins that determine the function of cells, organs, and the whole organism

• Current estimates are that there are 25,000 genes in the Human Genome

Proteins and Genes

• Many genes can lead to the production of multiple proteins

• Most genes produce proteins from both copies (located on each autosome)

• Sometimes only 1 copy is active – called genomic imprinting

DNA to RNA to Protein• Transcription vs. translation

• Types of RNA

• Differences from DNA– Ribose– Uracil

• Locations (in eukaryotes)

DNA to RNA to Protein

• Introns – noncoding region, not found in mature RNA

• Exons – the portion of DNA that determines the amino acid sequence

• Most genes in the human genome have at least one intron segment

DNA to RNA to Protein

• Gene = a sequence of DNA in the genome that is required for production of a functional product (polypeptide or RNA molecule)

• Also includes adjacent nucleotide sequences necessary for the proper expression (i.e. production of mRNA at correct time, in correct amount, and in the correct place)

DNA to RNA to Protein• Adjacent nucleotide sequence include ‘start’ and ‘stop’

instructions• At the 5’ end is a promoter region which initiates transcription• At the 3’ end is an untranslated region that causes a

sequence of adenosines to be added to the end of mature mRNA (called the polyA tail)

Gene Families• Code for closely related proteins

• i.e. hemoglobin

proteins are

made by genes

located close

together on

chromosomes

11 and 16

Pseudogenes

• DNA sequences that resemble known genes, but are nonfunctional– Nonprocessed – thought to be byproducts of

evolution, dead genes, used to be functional, now vestigial

– Processed – formed by retrotransposition, DNA to mRNA back to DNA, inserted into genome, lack introns, can be located random places

Transcription

• Genes are transcribed from the 3’ to 5’ strand of DNA

• They transcribe in the 5’ to 3’ direction, and include introns and exons

• A chemical cap is added to the 5’ end of the RNA, and the 3’ end is cleaved

• A polyA tail is added (which appears to increase stability)

• Then the RNA is processed and spliced to remove introns

Transport• This matured mRNA is transported out of

the nucleus to the cytoplasm

• Here translation

occurs in the

ribosome, in the

5’ to 3’ direction

Translation

• Translation is carried out by tRNA molecules, which bring specific amino acids to be added to the polypeptide chain

• Codons – located on mRNA

• Amino acids – 20 different types, most specified by multiple codons

• There are 3 nonsense or stop codons

Translation

• Methionine is always the first amino acids and its codon is called the initiator codon (AUG)

• tRNA has anticodons

Post-translational Processing

• Many proteins are further processed after translation

• Can be combined with other chains

• Can me modified chemically (i.e. adding methyl groups)

• Can have parts cut off (many mitochondrial proteins)

• Can be chopped into smaller pieces (insulin)

Mitochondrial Gene Expression

• Specialized RNA polymerase encoded in the nuclear genome transcribes mitochondrial genome

• Has 2 promoter regions (one for each strand of circular genome)

• Each strand transcribed entirely

August 14:

Medical Genetics

• One of the main goals of medical genetics is to identify mutations that cause disease

• This helps in determination of diagnosis and management

Medical Genetics

• There are 2 major problems that geneticists face– Getting enough DNA or RNA– Purifying the portion they want to analyze

Molecular Cloning

• Transfer of a DNA sequence into a microorganism

• Culturing of this microorganism

• These are clones, all containing the added DNA

• This process is known as molecular cloning

Restriction Enzymes• Discovered in the 1970’s

• Bacterial restriction endonucleases

• Recognize specific DNA sequences and can be used to cleave double stranded DNA at certain locations

Restriction Enzymes

• Often palindromes

• Can be put back together with any other segment due to matching overhangs with DNA ligase

• Called a recombinant DNA molecule

Vectors

• DNA molecule that can replicate autonomously in a host

• This allows it to be made in large quantities

• This is called recombinant DNA technology

• Most common vector is the plasmid

Plasmid• Circular double stranded DNA molecules

that are located in bacteria or yeast cells

• Replicated independently from organism’s chromosomes

• Found naturally in bacteria– Carried resistance genes– Form of sexual gene transfer/reproduction

Libraries

• Collection of clones with different inserted DNA fragments

• Typically every segment of a genome is represented on at least one fragment

Nucleic Acid Analysis

• The difficulty in analyzing DNA is locating a specific sequence

• After digestion by restriction enzymes there can be millions of different fragments

• Typically gel electrophoresis is performed to isolate the molecules by size

• Then nucleic acid hybridization is done to identify the specific desired molecule

Southern Blotting

• A DNA sample is obtained

• It is digested by restriction enzymes

• Then the sample is added to wells in a gel

• Then an electric field is generated, and smaller fragments of DNA move faster then larger fragments, so the sample is sorted by size

• Resembles a smear in the gel

Southern Blotting

• The smear of DNA is denatured – typically with a strong base - forming single stranded molecules

• Then the sample is transferred to filter paper by blotting

• A labeled single stranded probe is added

• X ray film is then used to view the molecule

Allele Specific Oligonucleotide

• More specific than Southern Blot

• Smaller and can detect single base pair mismatches

• Can be used for known mutations (sickle cell and cystic fibrosis)

• Can be used for familial mutations

Northern Blotting

• Also called RNA blotting

• Used to determine the quantity and size of mRNA molecules

• Same essential procedure as the Southern Blot

• Not used much anymore

• Replaced by PCR based techniques

Polymerase Chain Reaction

• Known as PCR

• Alternative to cloning

• Can generate unlimited amounts of a DNA sequence

• Fast, cheap, efficient

• Sequence located between 2 oligonucleotide primers

• Enzymatic amplification

Polymerase Chain Reaction

• Exponential replication, because the new strands formed can be used to form more new strands

• Several billion copies of a DNA molecule can be generated in a few hours

Polymerase Chain Reaction

• Very small amounts of DNA are still sufficient

• Buccal rinse, 1 cell from a 3 day old embryo, sperm obtained from a rape victim, drops of dried blood

August 19:

Sanger Sequencing

• Most widely used technique for DNA sequence analysis

• Named after Fred Sanger

• Won Nobel Prize in 1980 for developing DNA sequencing, with Walter Gilbert

Sanger Sequencing• Basically any segment of DNA can now be

sequanced• Uses chemical analogues of the 4 bases• Called dideoxy nucleutides

– ddA– ddG– ddT– ddC

• Stop the DNA polymerase from attaching to the next strand

Sanger Sequencing

• This essentially stops the DNA strand at different points

• Each analogue is labeled with a different fluorescent dye

• The strand fragments are then arranged by size, and the dye markers can be read in order

Sanger Sequencing

• It is essential to know the sequence of a gene to determine ASO probes or PCR primers to use in diagnosis procedures

• This method was used to sequence the 3 billion base pairs in the human genome

Sanger Sequencing

• Also used to sequence E. coli, Saccharomyces cerevisiae yeast, Plasmodium falciparum, Anopheles mosquito, Caenorhabditis elegans worm, Drosophilia melanogaster, various fish, the chicken, the mouse, the rat, the chimpanzee, and many more organism’s genomes

Sanger Sequencing

• Extremely useful in evolutionary biology, and used to compare different organisms phylogenetically

Digital Image Capture

• Now better technology allows the capture of fluorescence across a microscopic field

• Also use of microarray is improving diagnosis

Fluorescent In Situ Hybridization

• FISH• Fluorescent dyes can

also be used to view whole chromosomes and chromosome abnormalities

• The fluorescent markers are called ‘chromosome painting probes’

Spectral Karyotyping

• Can use 24 different probes and view each of the 24 chromosomes

• This is called SKY

• Makes each chromosome ‘glow’ a different color

• Makes it very obvious when parts of chromosomes are mislocated

Comparative Genome Hybridization

• CGH• Used with microarrays to analyze small

segments of DNA and their abundance• Typically control is marked with green

marker, and patient DNA is marked with red marker

• Microarray is flooded• Ratio of red to green shows relative

abundance in patient’s DNA

Comparative Genome Hybridization

• Very useful in certain cancer genes

RNA Expression Arrays

• More general analysis tool

• Can create a ‘finger print’ that is individualized, but not specifically informative about which RNA sequences are more abundant– Called molecular phenotypes– Can be used to characterize various disease

states

RNA Expression Arrays

• Currently being used by oncologists to differentiate likely prognosis of similar appearing tumors

Western Blot

• Analysis of proteins encoded by normal and/or mutant genes

• Proteins are isolated from a cell extract

• They are then sorted by size– Possibly by gel electrophoresis

• Transferred to a membrane

Western Blot

• Membrane is incubated with antibodies

• A second antibody with fluorescent tag is then added

• This allows for viewing of whether a specific protein is present in a sample

• Useful in certain diseases like Duchenne or Becker Muscular Dystrophy