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Green with envy?? Jelly fish “GFP” Transformed vertebrates Gene Cloning and Karyotyping Gene Cloning • Techniques for gene cloning enable scientists to prepare multiple identical copies of genesized pieces of DNA. • Most methods for cloning pieces of DNA share certain general features. – For example, a foreign gene is inserted into a bacterial plasmid and this recombinant DNA molecule is returned to a bacterial cell. – Every time this cell reproduces, the recombinant plasmid is replicated as well and passed on to its descendents. – Under suitable conditions, the bacterial clone will make the protein encoded by the foreign gene. • One goal may be to produce a protein product for use. • A second goal may be to prepare many copies of the gene itself. – This may enable scientists to determine the gene’s nucleotide sequence or provide an organism with a new metabolic capability by transferring a gene from another organism. Restriction Enzymes • In nature, bacteria use restriction enzymes to cut foreign DNA, such as from phages or other bacteria. • Most restrictions enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific point in these sequences. • Each restriction enzyme cleaves a specific sequence of bases called a restriction site. – These are often a symmetrical series of four to eight bases on both strands running in opposite directions. – If the restriction site on one strand is 3’-CTTAAG-5’, the complementary strand is 5’-GAATTC-3 • Restriction enzymes cut covalent phosphodiester bonds of both strands, often in a staggered way creating single-stranded ends, sticky ends. – These extensions will form hydrogen-bonded base pairs with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme Recombinant DNA vectors • Recombinant plasmids are produced by splicing restriction fragments from foreign DNA into plasmids. – A plasmid is a circular piece of DNA found in bacteria and contain genes. – Plasmids can be used to insert DNA from another organism into a bacterial cell. • Then, as a bacterium carrying a recombinant plasmid reproduces, the plasmid replicates within it. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings • The process of cloning a gene in a bacterial plasmid can be divided into five steps. Blue colonies White colonies Fig. 20.3 Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings The polymerase chain reaction (PCR) clones DNA entirely in vitro • When the source of DNA is scanty or impure, the polymerase chain reaction (PCR) is quicker and more selective. Its limitation is that PCR only produces short DNA segments within a gene and not entire genes. • This technique can quickly amplify any piece of DNA without using cells. • Devised in 1985, PCR has had a major impact on biological research and technology. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings • The DNA is incubated in a test tube with special DNA polymerase, a supply of nucleotides, and short pieces of singlestranded DNA as a primer. Fig. 20.7 Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings PCR • PCR can make billions of copies of a targeted DNA segment in a few hours. – This is faster than cloning via recombinant bacteria. • In PCR, a three-step cycle: heating, cooling, and replication, brings about a chain reaction that produces an exponentially growing population of DNA molecules. – PCR can make many copies of a specific gene before cloning in cells, simplifying the task of finding a clone with that gene. – PCR is so specific and powerful that only minute amounts of DNA need be present in the starting material Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings • PCR has amplified DNA from a variety of sources: – fragments of ancient DNA from a 40,000-yearold frozen wooly mammoth, – DNA from tiny amount of blood or semen found at the scenes of violent crimes, – DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders, – DNA of viral genes from cells infected with difficult-to-detect viruses such as HIV. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Chromosomal abnormalities • Incorrect number of chromosomes – Nondisjunction • chromosomes don’t separate properly during meiosis – Chromosome mutations • Deletion • Inversion • Duplication • Translocation Nondisjunction • Problems with meiotic spindle cause errors in daughter cells – homologous chromosomes do not separate properly during Meiosis 1 – sister chromatids fail to separate during Meiosis 2 – too many or too few chromosomes 2n n-1 n n+1 n Alteration of chromosome number Nondisjunction • Baby has wrong chromosome number – Trisomy • cells have 3 copies of a chromosome – Monosomy • cells have only 1 copy of a chromosome n+1 n-1 n n trisomy monosomy 2n+1 2n-1 Human chromosome disorders • High frequency in humans – most embryos are spontaneously aborted – alterations are too disastrous – developmental problems result from biochemical imbalance • imbalance in regulatory molecules? – hormones? – transcription factors? • Certain conditions are tolerated – upset the balance less = survivable – but characteristic set of symptoms = syndrome Genetics Laboratory • Cytogenetics Tissue culture Harvesting/Slide Preparation FISH Analysis Karyotyping Results / Interpretation Report Genetic testing • Amniocentesis in 2nd trimester – sample of embryo cells – stain & photograph chromosomes • Analysis of karyotype Karyotyping Chromosome Spread Karyotype of a normal male Down syndrome • Trisomy 21 – 3 copies of chromosome 21 – 1 in 700 children born in U.S. • Chromosome 21 is the smallest human chromosome – but still severe effects • Frequency of Down syndrome correlates with the age of the mother Down syndrome & age of mother Mother’s age Incidence of Down Syndrome Under 30 <1 in 1000 30 1 in 900 35 1 in 400 36 1 in 300 37 1 in 230 38 1 in 180 39 1 in 135 40 1 in 105 42 1 in 60 44 1 in 35 46 1 in 20 48 1 in 16 49 1 in 12 Rate of miscarriage due to amniocentesis: 1970s data 0.5%, or 1 in 200 pregnancies 2006 data <0.1%, or 1 in 1600 pregnancies Sex chromosomes abnormalities • Human development more tolerant of wrong numbers in sex chromosome • But produces a variety of distinct syndromes in humans – – – – XXY = Klinefelter’s syndrome male XXX = Trisomy X female XYY = Jacob’s syndrome male XO = Turner syndrome female Klinefelter’s syndrome • 2X and 1Y – one in every 2000 live births – have male sex organs, but are sterile – feminine characteristics • some breast development • lack of facial hair – tall – normal intelligence Klinefelter’s syndrome Jacob’s syndrome male • 1X and 2 Y – 1 in 1000 live male births – extra Y chromosome – slightly taller than average – more active – normal intelligence, slight learning disabilities – delayed emotional immaturity – normal sexual development Trisomy X • 3X – 1 in every 2000 live births – produces healthy females • Why? • all but one X chromosome is inactivated Turner syndrome • 1X – 1 in every 5000 births – varied degree of effects – webbed neck – short stature – sterile replication crossing over error of error of Changes in chromosome structure • Deletion – loss of a chromosomal segment • Duplication – repeat a segment • Inversion – reverses a segment • Translocation – move segment from one chromosome to another Translocation 46,XY,t(8;9)(q24.3;q22.1) FISH analysis: abl/bcr Genes on Diploid Cells and Ph Positive Translocation CML Cells Normal Dosage Compensation Do males have half as much of the products of genes on the X as do females? NO!! X Inactivation Barr Body: Inactive X Interphase: Chromomes can’t be stained, but a dark-staining body is visible in the nuclei of cells of female mammals Which X gets inactivated? Mary Lyon & Lianne Russell (1961) proposed that one or other of X becomes inactivated at a particular time in early development. Within each cell,which X becomes inactivated is random. As development proceeds, all cells arising by cell division after that time have same X inactivated. In 64-cell embryos Adult female mammals have two copies of each gene on the X chromosome.