Gene Cloning and Karyotyping

Gene Cloning Techniques for gene cloning enable scientists to prepare multiple identical copies of gene- sized 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 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. Recombinant DNA vectors 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. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Fig. 20.3

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 n n-1 n+1

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+1n n-1n monosomy 2n-1 trisomy 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

Karyotyping Karyotype of a normal male Chromosome Spread

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 in in in in in in in in in in in 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

Genetic testing Amniocentesis in 2nd trimester –sample of embryo cells –stain & photograph chromosomes Analysis of karyotype

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

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 3 X –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

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 error of replication error of crossing over

46,XY,t(8;9)(q24.3;q22.1)