Genome Project - Primer on molecular genetics - 1992 (522926), страница 2
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Allliving organisms are composed largely of proteins; humans can synthesize at least100,000 different kinds. Proteins are large, complex molecules made up of long chains ofsubunits called amino acids. Twenty different kinds of amino acids are usually found inproteins. Within the gene, each specific sequence of three DNA bases (codons) directsthe cell’s protein-synthesizing machinery to add specific amino acids.
For example, thebase sequence ATG codes for the amino acid methionine. Since 3 bases code for1 amino acid, the protein coded by an average-sized gene (3000 bp) will contain 1000amino acids. The genetic code is thus a series of codons that specify which amino acidsare required to make up specific proteins.The protein-coding instructions from the genes are transmitted indirectly through messenger ribonucleic acid (mRNA), a transient intermediary molecule similar to a single strandof DNA. For the information within a gene to be expressed, a complementary RNA strandis produced (a process called transcription) from the DNA template in the nucleus. ThisComparative Sequence SizesLargest known continuous DNA sequence(yeast chromosome 3)Escherichia coli (bacterium) genomeLargest yeast chromosome now mappedEntire yeast genomeSmallest human chromosome (Y)Largest human chromosome (1)Entire human genomeBases350 Thousand4.65.815502503MillionMillionMillionMillionMillionBillionFig.
3. Comparison of Largest Known DNA Sequence with Approximate Chromosome andGenome Sizes of Model Organisms and Humans. A major focus of the Human Genome Projectis the development of sequencing schemes that are faster and more economical.7Primer onMolecularGeneticsmRNA is moved from the nucleus to the cellular cytoplasm, where it serves as the template for protein synthesis.
The cell’s protein-synthesizing machinery then translates thecodons into a string of amino acids that will constitute the protein molecule for which itcodes (Fig. 5). In the laboratory, the mRNA molecule can be isolated and used as atemplate to synthesize a complementary DNA (cDNA) strand, which can then be used tolocate the corresponding genes on a chromosome map. The utility of this strategy isdescribed in the section on physical mapping.ChromosomesThe 3 billion bp in the human genome are organized into 24 distinct, physically separatemicroscopic units called chromosomes. All genes are arranged linearly along the chromosomes.
The nucleus of most human cells contains 2 sets of chromosomes, 1 set given byeach parent. Each set has 23 single chromosomes—22 autosomes and an X or Y sexchromosome. (A normal female will have a pair of X chromosomes; a male will have an XORNL-DWG 91M-17361DNA ReplicationA TCGT AGCA TCGParentStrandsT AGCFig. 4. DNA Replication.During replication the DNAmolecule unwinds, witheach single strandbecoming a template forsynthesis of a new,complementary strand.Each daughter molecule,consisting of one old andone new DNA strand, is anexact copy of the parentmolecule.
[Source:adapted from Mapping OurGenes—The GenomeProjects: How Big, HowFast? U.S. Congress,Office of TechnologyAssessment, OTA-BA-373(Washington, D.C.: U.S.Government PrintingOffice, 1988).]8A TCGT AGCA TCGT AGCATCTG CA TCGT AAGCACA TCGTGGCComplementaryNew StrandGAT AGCTGA TCGA TCGT AG CA TCGT AGCT AGCA TCGT AGCA TA TComplementaryNew Strandand Y pair.) Chromosomes contain roughly equal parts of protein and DNA; chromosomalDNA contains an average of 150 million bases. DNA molecules are among the largestmolecules now known.Chromosomes can be seen under a light microscope and, when stained with certain dyes,reveal a pattern of light and dark bands reflecting regional variations in the amounts of Aand T vs G and C.
Differences in size and banding pattern allow the 24 chromosomes tobe distinguished from each other, an analysis called a karyotype. A few types of majorchromosomal abnormalities, including missing or extra copies of a chromosome or grossbreaks and rejoinings (translocations), can be detected by microscopic examination;Down’s syndrome, in which an individual's cells contain a third copy of chromosome 21, isdiagnosed by karyotype analysis (Fig.
6). Most changes in DNA, however, are too subtle tobe detected by this technique and require molecular analysis. These subtle DNA abnormalities (mutations) are responsible for many inherited diseases such as cystic fibrosis andsickle cell anemia or may predispose an individual to cancer, major psychiatric illnesses,and other complex diseases.ORNL-DWG 91M-17360NUCLEUSGeneDNAFree Amino AcidsmRNACopyingDNA inNucleustRNA BringingAmino Acid toRibosomeGrowingProtein ChainAminoAcidsmRNARIBOSOME incorporatingamino acids into thegrowing protein chainmRNACYTOPLASMFig. 5.
Gene Expression. When genes are expressed, the genetic information (base sequence) on DNA is first transcribed(copied) to a molecule of messenger RNA in a process similar to DNA replication. The mRNA molecules then leave the cellnucleus and enter the cytoplasm, where triplets of bases (codons) forming the genetic code specify the particular amino acids thatmake up an individual protein. This process, called translation, is accomplished by ribosomes (cellular components composed ofproteins and another class of RNA) that read the genetic code from the mRNA, and transfer RNAs (tRNAs) that transport aminoacids to the ribosomes for attachment to the growing protein.
(Source: see Fig. 4.)9Primer onMolecularGeneticsFig. 6. Karyotype. Microscopic examination of chromosome size and banding patterns allowsmedical laboratories to identify and arrange each of the 24 different chromosomes (22 pairs ofautosomes and one pair of sex chromosomes) into a karyotype, which then serves as a tool in thediagnosis of genetic diseases. The extra copy of chromosome 21 in this karyotype identifies thisindividual as having Down’s syndrome.Mapping and Sequencing the Human GenomeA primary goal of the Human Genome Project is to make a series of descriptive diagrams—maps—of each human chromosome at increasingly finer resolutions.
Mappinginvolves (1) dividing the chromosomes into smaller fragments that can be propagated andchar-acterized and (2) ordering (mapping) them to correspond to their respective locationson the chromosomes. After mapping is completed, the next step is to determine the basesequence of each of the ordered DNA fragments.
The ultimate goal of genome research isto find all the genes in the DNA sequence and to develop tools for using this information inthe study of human biology and medicine. Improving the instrumentation and techniquesrequired for mapping and sequencing—a major focus of the genome project—will increase efficiency and cost-effectiveness. Goals include automating methods and optimizing techniques to extract the maximum useful information from maps and sequences.A genome map describes the order of genes or other markers and the spacing betweenthem on each chromosome. Human genome maps are constructed on several differentscales or levels of resolution. At the coarsest resolution are genetic linkage maps, whichdepict the relative chromosomal locations of DNA markers (genes and other identifiableDNA sequences) by their patterns of inheritance.
Physical maps describe the chemicalcharacteristics of the DNA molecule itself.10Geneticists have already charted the approximate positions of over 2300 genes, and astart has been made in establishing high-resolution maps of the genome (Fig. 7). Moreprecise maps are needed to organize systematic sequencing efforts and plan newresearch directions.Mapping StrategiesGenetic Linkage MapsA genetic linkage map shows the relative locations of specific DNA markers along thechromosome. Any inherited physical or molecular characteristic that differs among individuals and is easily detectable in the laboratory is a potential genetic marker. Markerscan be expressed DNA regions (genes) or DNA segments that have no known codingfunction but whose inheritance pattern can be followed. DNA sequence differences areespecially useful markers because they are plentiful and easy to characterize precisely.ORNL-DWG 91M-17362ANUMBER OF EXPRESSED GENES MAPPED2500Fig.
7. Assignment of Genesto Specific Chromosomes.The number of genes assigned(mapped) to specific chromosomes has greatly increased sincethe first autosomal (i.e., not on theX or Y chromosome) marker wasmapped in 1968. Most of thesegenes have been mapped tospecific bands on chromosomes.The acceleration of chromosomeassignments is due to (1) a combination of improved and newtechniques in chromosome sortingand band analysis, (2) data fromfamily studies, and (3) the introduction of recombinant DNAtechnology.
[Source: adapted fromVictor A. McKusick, “CurrentTrends in Mapping HumanGenes,” The FASEB Journal 5(1),12 (1991).]200015001000500066 68 70 72 74 76 78 80 82 84 86 88 90YEAR9211Primer onMolecularGeneticsMarkers must be polymorphic to be useful in mapping; that is, alternative forms must existamong individuals so that they are detectable among different members in family studies.Polymorphisms are variations in DNA sequence that occur on average once every 300 to500 bp. Variations within exon sequences can lead to observable changes, such as differences in eye color, blood type, and disease susceptibility. Most variations occur withinintrons and have little or no effect on an organism’s appearance or function, yet they aredetectable at the DNA level and can be used as markers.
Examples of these types ofmarkers include (1) restriction fragment length polymorphisms (RFLPs), which reflectsequence variations in DNA sites that can be cleaved by DNA restriction enzymes (seebox), and (2) variable number of tandem repeat sequences, which are short repeatedsequences that vary in the number of repeated units and, therefore, in length (a characteristic easily measured). The human genetic linkage map is constructed by observing howfrequently two markers are inherited together.Two markers located near each other on the same chromosome will tend to be passedtogether from parent to child.
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