AP Biology CMR @ SOHI ‘98 B.Rife page 1 / 7

IV. Unit 4: The Human Genome - Chapter 13 I. Introduction Mendelian and molecular genetics are powerful tools that provide humans with a means for understanding and predicting the consequences of mating.

The added power of recombinant DNA technology promises the deciphering of the human genome and uncovering the molecular causes of many genetic afflictions.

II. Physical description of the genome 13.1 Three billion nucleotide pairs are packed into the human genome The human genome is all the genes present in a haploid human cell. The total length of DNA in a diploid cell (with 6 billion nucleotide pairs) is 3 m, packed into 46 chromosomes. Although the amount of DNA in a human genome is 1000 times that in E. coli, and E. coli has about 2000 genes, the human genome probably only has between 50,000 and 100,000 genes (that is, 25-50 times the number of genes) This is because about 80% of the human genome is noncoding: introns, regulatory sequences, and structural sequences.

13.2 A karyotype is a photographic inventory of an individual’s chromosomes Blood samples are cultured for several days under conditions that promote cell division of white blood cells. (Red blood cells lack nuclei and do not divide.) The culture is treated with a chemical that stops cell division at metaphase. White blood cells are separated, stained, and squashed (to spread out the chromosomes) following the procedure. The individual chromosomes in a photograph are cut out and rearranged by number. A normal human karyotype shows twenty-two pairs of autosomes and one pair of sex chromosomes. Males have an X and a Y chromosomes; females have two X chromosomes

III. Genetic disorders due to abnormalities in chromosome number.

13.3 An extra copy of chromosome 21 causes Down syndrome In most cases, human offspring that develop from zygotes with an incorrect number of chromosomes abort spontaneously. Trisomy 21 is the most common chromosome-number abnormality, occurring in about one out of 700 births. Down syndrome includes a number and range of physical, mental, and disease-susceptibility features. AP Biology CMR @ SOHI ‘98 B.Rife page 2 / 7

The incidence of Down syndrome increases with the age of the mother. The age of the father shows a slight correlation with the increased incidence of Down syndrome.

13.4 Accidents during meiosis can alter chromosome number Nondisjunction is the failure of chromosome pairs to separate during either meiosis I or meiosis II. Nondisjunction causes an abnormal chromosomal number in the gametes. Offspring inherit an extra chromosome (trisomy) or are missing a chromosome (monosomy)

Fertilization of an egg resulting from nondisjunction with a normal sperm results in a zygote with abnormal chromosome number. The explanation for the increased incidence of trisomy 21 among older women is not entirely clear but probably involves the length of time a woman’s developing eggs are in meiosis. Meiosis begins in all eggs before the woman is born, and finishes as each egg matures in the monthly cycle following puberty. Eggs of older woman have been “within” meiosis longer.

13.5 Nondisjunction can alter the number of sex chromosomes Unusual numbers of sex chromosomes upset the genetic balance less than do unusual numbers of autosomes, perhaps because the Y chromosome carries fewer genes and extra X chromosomes are inactivated as Barr bodies in females. Abnormalities in sex chromosome number result in individuals with a variety of different characteristics, some more seriously affecting fertility or intelligence than others. These sex chromosome abnormalities illustrate the crucial role of the Y chromosome in determining a person’s sex. A single Y is enough to produce “maleness” even in combination with a number of Xs, whereas the lack of a Y results in “femaleness”

One sex chromosome monosomy (Turner syndrome XO) and three trisomies (Klinefelter syndrome XXY, metafemale XXX, and XYY male) are known in human beings.

IV. Genetic disorders due to abnormalities in chromosome structure.

13.6 Alterations of chromosome structure may cause serious disorders Deletions, the lost of a chromosomal fragment, duplication, the joining of an additional chromosomal fragment, and inversion, the reversal of a chromosomal fragment occur within one chromosome.

Inversions are less likely to produce harmful effects than deletions or duplications because all the chromosome’s genes are still present. AP Biology CMR @ SOHI ‘98 B.Rife page 3 / 7

Duplications, if they result in the duplication of an ongogene in somatic cells, may increase the incidence of cancer.

Translocation involves the transfer of a chromosome fragment between nonhomologous chromosomes. Translocations may not be harmful, but they may be associated with increases in cancer, if the translocated segment activates an oncogene in a somatic cell.

13.7 Pedigrees track genetic traits through a family history A pedigree is the ancestral line of an individual. By applying Mendel’s principles, one can deduce the information on the chart from the pattern of phenotypes.

13.8 Many human disorders are inherited as Mendelian traits Over 1000 known genetic traits are attributable to a single gene locus and show simple Mendelian patterns of inheritance. Most disorders are caused by recessive alleles and vary in the severity of the expressed trait. The vast majority of people afflicted with recessive disorders are born to normal, heterozygous parents (carriers).

Cystic fibrosis is the most common lethal genetic disease in the U.S.

Most genetic diseases of this sort are not evenly distributed across all racial and cultural groups because of the prior and existing reproductive isolation of various populations. (sickle-cell anemia)

Sickle-cell disease is an example of a human genetic disorder that is controlled by incompletely dominant alleles. (HbA HbA - normal, HbS HbS - sickle-cell disease, HbA HbS - sickle-cell trait) Laws forbidding inbreeding may have arisen from observations that such marriages more often resulted in miscarriages, stillbirths, and birth defects.

Some disorders are caused by dominant alleles. These disorders vary in how deadly they are. Some are nonlethal handicaps, some are lethal in the homozygous condition, and some are intermediate in severity. Achondroplasia, a type of dwarfism, is lethal in the homozygous condition; individuals who express the trait are heterozygous. Other conditions attributable to dominant alleles are lethal only in older adults, so the allele can be passed to children before it is realized that the parent has the condition. (Huntington’s disease) AP Biology CMR @ SOHI ‘98 B.Rife page 4 / 7

13.9 Sex-linked disorders affect mostly males Most known sex-linked traits are caused by genes (alleles) on the X chromosome. When these traits are recessive (most are), males express them because they only have one X. Females who have the allele are normally carriers and will exhibit the condition only if they are homozygous. Males cannot pass sex-linked traits to sons (who get a Y from their father). Red-green color blindness is a complex of sex-linked disorders, each of which is caused by an allele on the X chromosome. The result is considerable variation in the changes in color perception. Hemophilia is a sex-linked trait with a particularly well-studied history because of its incidence among the intermarrying royal families of Europe. Duchenne muscular dystrophy is a severe disease affecting muscle tissue and has been traced to a particular nucleotide sequence.

13.10 Genetic counselors can help prospective parents These staff members of many hospitals gather information (largely based on an analysis of family pedigrees) concerning a couple’s risk of having an affected child, and then counsel the prospective parents.

13.11 Fetal testing can spot many disorders early in pregnancy Amniocentesis involves removing amniotic fluid that bathes the fetus at 14-16 weeks. This fluid contains living fetal cells (from skin and mouth cavity) and can be karyotyped. Some chemical tests can be performed on the fluid itself. Chorionic villi sampling involves removing tissue from the fetal side of the placenta nurturing a fetus at 8-10 weeks. These cells are rapidly dividing and can be immediately karyotyped. Some biochemical tests can be performed. Ultrasound imaging of the fetus provides a noninvasive view inside the womb. Fetoscopy provides a more direct view of the fetus through a needle-thin viewing scope inserted into the uterus. Amniocentesis, chorionic villi sampling, and fetoscopy carry a small risk and are reserved for situations with higher probabilities of disorders (for example, older parents or situations where genetic counseling has uncovered a higher risk).

13.12 Carrier recognition is a key strategy in human genetics Most children with recessive genetic disorders (the most common type) are born to parents with normal phenotypes. A number of biochemical tests exist for identifying carriers of some genetic disorders. A method similar to DNA probing will likely provide many more diagnostic procedures to help recognize carriers. AP Biology CMR @ SOHI ‘98 B.Rife page 5 / 7

VI. Some recent developments in dealing with genetic disorders. 13.13 Nancy Wexler: A top researcher discusses a mind-ravaging genetic disease American human geneticist Nancy Wexler has chosen to work on Huntington’s disease (HD) because her mother had HD and she has a 50% chance of contracting the disease.

HD is characterized by a slow deterioration of the central nervous system. In 1983, she and her associates have determined a pedigree for 10,00 Venezuelan people in several villages where the incidence of HD is high. In 1993, by comparing the inheritance pattern of this disease with that of other traits known to be caused by genes on various chromosomes, they determined that the gene was on chromosome 4. The HD gene is a so-called expanding gene, containing a short nucleotide sequence (CAG) that is repeated from 11 to over 100 times. A normal allele has 11-34 repeats, while the disease allele has none. Scientists are currently attempting to show what the normal allele does and what the molecular basis of the disease is, the ultimate goal being a treatment or cure for the disease.

13.14 Restriction fragments analysis is a powerful method for carrier detection Nucleotide sequences of all but identical twins are different. Extracted DNA from a person’s cells can be cut up into a set of fragments by reacting the DNA with a series of different restriction enzymes. Sets of these fragments (restriction fragment length polymorphisms, or RFLPs) differ in length and number between different, nonidentical twin individuals.

These DNA fragments are positively charged, and different DNA lengths will migrate different distances toward a negative charge in an eletrophoretic gel. The process of separating fragments according to their lengths is called gel electrophoresis.

The use of radioactive probes allows specific sequences to be separated from all the other fragments, and the resulting pattern to be recorded on X-ray film. The darkness of the developed spot is an indication of the amount of each fragment size. FRLP analysis was used to enable workers studying Huntington’s disease find a genetic marker (unique restriction sites) closely associated to the HD gene on chromosome 4. Once the marker is known for a particular disease, RFLP analysis can be used to test for it. Restriction fragment analysis requires about 1 µg of DNA. AP Biology CMR @ SOHI ‘98 B.Rife page 6 / 7

13.15 The PCR method is used to amplify DNA samples The polymerase chain reaction (PCR) is a technique for copying a single DNA sequence many times.

A mixture of the DNA, DNA polymerase, and nucleotide monomers will continue to replicate, forming a geometrically increasing number of copies. This technique has revolutionized DNA work because sequences can now be obtained from extremely small samples. Prehistoric DNA from a number of sites has been cloned into partial genomes in this way (prehistoric men, mammoths, and 30-million year-old plants, but not dinosaurs?).

VII. Uses for these techniques. 13.16 DNA technology and the law DNA fingerprinting uses RFLP analysis to identify small amounts of DNA from blood, tissue, or semen. PCR is not used to amplify smaller amounts because the technique is too sensitive and might amplify the wrong DNA. In criminal investigations, RFLPs are compared with those of other sources. In paternity cases, RFLPs from DNA fragments of a suspected parent are compared with those of the child and the other parent. DNA from a single sperm is enough to identify a suspected rapist when PCR amplification precedes DNA fingerprinting. (Mader 1996) These is some controversy surrounding this technique. One concern is that only a few regions of DNA are tested, not the entire genome (which would be prohibitively expensive). However, the regions chosen are known to vary widely between individuals, and the results can be stated in terms of probabilities.

13.17 Gene therapy is already a reality, and it raises serious ethical questions. In certain cases where a disorder is due to a single gene, it is possible to replace defective genes with normal genes. An example is a trial procedure that should cure individuals with an autosomal recessive allele that causes defective functioning of the immune system and is usually fatal. Bone marrow stem cells, which are essential for blood cell formation, are removed. By means of a retrovirus, the defective gene is replaced with the normal one. The recombinant cells are cloned in culture and reintroduced in the individual, after the natural bone marrow cells have all been killed.

Gene therapy is now a reality, and researchers are envisioning all sorts of applications aimed at curing human genetic disorders as well as many other types of illnesses. AP Biology CMR @ SOHI ‘98 B.Rife page 7 / 7

13.18 The Human Genome Project is a scientific adventure This international project proposes to completely sequence the human genome in four interlocking lines of work. The first will locate several thousand genetic markers spaced evenly along all chromosomes, to act as a set of references for other work. The second will map sequenceable fragments on each chromosome. The third will produce the exact order of the nucleotide pairs in each fragment and hence each chromosome. Finally, by comparing sequences in the human genome with those of other species, it should be possible to begin to define the functions of some of the sequences. Technical problems include how to rapidly sequence so many fragments and how to handle the huge amounts of data. The project has huge potential benefits: insight into embryonic development and evolution, and identification of genes that cause genetic disorders and genes that are partly implicated in more common diseases such as cancer, heart disease, diabetes, schizophrenia, alcoholism, and Alzheimer’s disease.

13.19 The ethical questions raised by human genetics demand our attention The Human Genome Project offers some potential for misuse of the information by governments, employers, and insurers, which we as citizens must be aware of and prevent. The possibility of gene therapy suggests a return to the notion of eugenics (the systematic removal of deleterious genes from the human population). Genetic discrimination by potential employers is a concern. The best way we can help control the use of these techniques and the information they result in is to become aware of how they work.