Medical Biotechnology lecture 4.p ptx

x

1. Molecular Genetics A large proportion of the genomes of higher eukaryotic organisms does not code for mRNAs or any other RNAs required by the organism More than 95% of human chromosomal DNA is in such state Non-coding regions are similar but not identical – reason why DNA fingerprinting can distinguish between individuals. Molecularly, a gene is the entire nucleic acid sequence that is necessary for the synthesis of a functional gene product

2. Most Eukaryotic Genes Produce Monocistronic mRNAs and contian lengthy introns . Cistron is a protein coding region Introns are non-coding regions and are taken out during RNA processing Simple and complex transcription units are found in eukaryotic genomes Primary transcript produced from simple transcription unit yields a single type of mRNA encoding a single protein. Affected by mutations

3. Comparative Density of Genes

4. Complex transcription units have primary RNA transcript processed in more than one way, leading to formation of mRNA containing different exons. Multiple mRNAs can arise from a primary transcript in three different ways Using different splice sites – have same 5’ and 3’ exons but different internal exons

5. Using alternative poly(A) sites – have same 5’ exons but different 3’ exons Using alternative promoters – different 5’ exons and common 3’ exons

6. Chromosomal Organization of Genes and Noncoding DNA Genomes of many organisms contain lots of non-functional DNA Aprox . One third of human genomic DNA is thought to be transcribed into pre-mRNA. Some 95% of these sequences are in introns which are removed by RNA splicing Protein-Coding genes may be solitary or belong to a gene family

7. 25-50% of protein coding genes in multicellular organisms are represented only once – solitary genes eg chicken lysozyme gene Duplicated genes encode proteins with similar but non-identical AA sequences – protein families eg 70-kDa heat-shock proteins

8. Tandemly repeated genes encode rRNAs , tRNAs , and Histones In tandenmly repeated genes, copies of a sequence appear one after the other in a head to tail fashion over a long stretch of DNA Needed to meet cellular requirements for their transcripts

9. Most simple-sequence DNA are concentrated in specific chromosomal locations Simple-sequence commonly refered to as satellite DNA. In case of repeats contiaining 1-13 base pairs – microsatellites Microsatellites – thought to have originated by “backward slippage” of daughter strands on its template strand during replication Some come from slippage in germ cells and such microsatellites cause at least 14 different types of neuromascular diseases

10. Satellite DNA lies near centromeres while some can be found at telomeres Others can be at specific locations within chromosome arms thereby allowing identification of particular chromosomes by fluorescence in situ hybridization (FISH)

11. Simple-sequence DNAs are useful chromosomal markers FISH: hybridization in situ with a particular Simple-sequence DNA labeled with a fluorescent biotin and viewed under appropriate wavelength of light

12. DNA fingerprinting depends on differences in length of simple-sequence DNAs Differences in the number of repeats and thus the length of simple-sequence tandem repeats thought to arise from unequal crossing over within regions during meiosis This gives unique arrays in each individual These polymorphisms form the basis of DNA fingerprinting for identifying individuals.

13. Mobile DNA (Transposable Elements) Interspersed repeats aka moderately repeated DNA or intermediate-repeat DNA have unique ability to move in the genome Discovered by Barbara McClintock in maize during the 1940s Sometimes referred to as “selfish DNA” Transposition – process of copying and inserting into new sites Transposition involves a DNA (DNA transposon ) or an RNA intermediate where reverse transcription is used ( retrotransposons ).

14. Transposition

15. Some retro- transposons contain long terminal repeats (LTRs) and behave like retroviruses Common in yeast (e.g. Ty elements) and drosophila (e.g. copia elements) Less abundant in mammals Sometimes called viral retro- transposons due to their similarities with retroviruses

16. Eukaryotic Chromosomes Eukaryotic nuclear DNA associates with histone proteins to form chromatin Chromatin exists in extended (at low salt conc.) and condensed (at physiological salt conc.) forms Modifications ( eg acetylation on H3 and H4) of histone tails controls chromotin condensation Nonehistone proteins provide structural scaffold for long chromatin loops

17. Chromatin contains small amounts of other proteins ( eg DNA binding transcription factors) in addition to histones and scaffold proteins Eukaryotic chromosomes contain one linear DNA molecule

18. Morphology and functional elements of Eukaryotic Chromosomes Chromosome number, size, and shape at metaphase are species-specific During metaphase, chromosomes can be distinguished by banding patterns and chromosome painting Mild heating and staining with giemsa reagent produces G bands Treating with hot alkaline solution before giemsa staining produces R bands Chromosome painting – a variation of FISH – uses probes specific for sites scattered along the length of each chromosome differentiating between similar sizes and shapes.

19. Multicolor FISH can detect chromosomal translocations Interphase polytene chromosomes arise by DNA amplification Drosophila species contain polytene chromosomes as a result of DNA amplification that can be visualized by fixing and staining. Heterochromatin consists of chromosome regions that do not uncoil