Identification of genetic material


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Identification of genetic material

  1. 1. GENETICSDEFINITIONGenetics is the science that seeks tounderstand, explain, and ultimately exploitthe phenomenon of heredity (i.e. thetransmission of biological characteristics oforganisms from one generation to the next –from parents to offspring).
  2. 2. HISTORICAL BACKGROUNDThe superficial facts of heredity have been known to mankind since the prehistoric time, hence the old-age saying, “like begets like”.
  3. 3. Pondering on the intricacy of the hereditary phenomenon one early naturalist had this to say,“It often happens also that the children may appear like a grandfather and reproduce the looks of a great grand-parent, because the parents often conceal in their bodies many primordial mingled in many ways, which fathers hand on to fathers received from their stock; from these Venus brings forth forms with varying lot, and reproduce the countenance, the voice, the hair of their ancestors
  4. 4. However the mechanism of thephenomenon has eluded the inquisitivemind of man for ages until the basic factsof biology, particularly those concernedwith reproduction became knowntowards the end of the nineteenthcentury and beginning of the twentiethcentury.
  5. 5. Up until late 19th century work on the genetic phenomenon had been investigated along two main lines of approach, namely: the identification of the physical nature of the genetic material the understanding of the manner by which biological characters are inherited.
  6. 6. Since before the dawn of the 20th centuryvery little was known about eitherphenomenon these lines of investigationwere pursued separately.
  7. 7. IDENTIFICATION OF GENETICMATERIAL In the very early days of investigation of the genetic phenomenon it was thought that biological materials could arise spontaneously from decaying matter. However this belief was later disproved through a series of experiments in which it was shown that no organisms developed in sealed flasks containing boiled organic matter.
  8. 8. Latter, with the development of thescience of classification of biologicalorganisms into separate and distinctspecies it became evident that organismsof one species could only give rise toorganisms of the same type.
  9. 9.  Since spontaneous generation of organisms implied non-fixity of species the theory was finally dropped in favour of the theory of continuity of life which propounded that new organisms arise only through the continuity of life.
  10. 10.  After the general acceptance of the concept of a continuous transfer of living material between generations several theories were proposed to explain how the transfer could be effected. It was generally accepted at least that an organism develops from a miniscule piece of transmitted matter, though the physical nature of the matter remained controversial.
  11. 11.  Some workers believed that an organism developed from a substance received from the female egg and a contribution of form received from the male seminal fluid. Latter, after the discovery of sex cells (i.e. the egg and sperm) it was theorized that one of the sexes contained within it the entire organism in perfect miniature form which could develop into its preformed adult proportions – the pre- formation.
  12. 12. However latter it was demonstrated thatdifferent adult structures develop fromuniform embryonic tissues which provideno clue as to their ultimate fate.
  13. 13. This led to the replacement of the pre-formation theory by another concept whichpropounded that tissues, organs, andmany other new factors which were notpresent in the original formation of anorganisms appeared in the course ofdevelopment of the organism.
  14. 14. The appearance of the organs was believed to arise entirely through mysterious vital forces - the epigenetic concept.
  15. 15. This view was later modified by theproposition that organs arose through agradual transformation of increasinglyspecialized tissue.
  16. 16.  It was latter advanced that an organism developed from very small invisible components (gemmules) which were exact copies of each body organ. These were thought to be transmitted by the blood stream to the sex organs where they assembled into gametes.
  17. 17.  Upon fertilization mixture of paternal and maternal organs and tissues would be constituted, followed by the distribution of the miniature elements to different parts of the body during development.
  18. 18. This theory was well received by believers of evolution since it provided an explanation of how heritable changes could occur, leading to the appearance of new species.
  19. 19. It was propounded that each of thesehereditary agents had a spiritual consciouslike property that could perceive andinterpret messages from the outside.
  20. 20.  Thus, for example, the excess use or disuse of an organ would change its gemmules and consequently lead to a changed inheritance in the descendants.
  21. 21. This theory was however quickly disproved by empirical experimentation – the work of John Lamark.
  22. 22. Instead it was advanced that multi-cellular organisms give rise to two types of tissue, i.e. somatic and germplasmic tissue.Somatic tissue was considered to be essential for life processes of the organism but was not capable of sexual reproduction. Thus changes occurring in somatic tissues could not be passed on in heredity.
  23. 23. On the other hand, germsplasmic tissue is capable of reproduction and hence any changes occurring within it could lead to altered inheritance.
  24. 24. The germplasm was considered to be transmitted from one generation to the next and accounted for the many biological similarities between ancestors and descendants.
  25. 25. An end to the early beliefs and theoriesconcerning the nature of hereditarymaterial approached with the advent ofmicroscope, and hence the consequentdetailed knowledge of cell structure andcell division.
  26. 26. At first the nucleus was shown to bedirectly involved in fertilization through theunion of the sperm and egg nuclei.
  27. 27. Latter dark staining nuclear threads (latternamed chromosomes) were shown todivide longitudinally during cell division,with passage of equal portions of thematerial to the two daughter cells
  28. 28. The total number of the threads was shownto be constant in each cell of an organismand species except for gametes whichcontained half the number of threads inother cells.
  29. 29. However, the number of nuclear threadswas shown to be restored when the nucleiof the gametes fused during fertilization toform the first embryonic cell.
  30. 30.  These observations provided the first clue regarding the transmission of hereditary factors from one generation to the next.
  31. 31.  Since the splitting of the parental chromosomes occurred longitudinally, and since the chromosomes of the offspring were equal to the number of parental chromosomes it was reasoned that the link between parents and offspring occurred through gametes.
  32. 32. TRANSMISSION OF GENETIC MATERIAL Meanwhile, in another scenario, themechanism of heredity was beinginvestigated by crossing different parentalstocks exhibiting contrasting visiblecharacteristics and observing the outcomein the offspring.
  33. 33.  Originally it had been long believed heredity was a blending process whereby the different parental characteristics blended with each other.
  34. 34.  This belief however was dismissed by observations that offspring often resembled one or the other parent.
  35. 35. This led to speculations that parents could contribute quantitatively differently to the inheritance of the offspring. The direction of the wind at fertilization was also believed to affect the transmission of heredity to offspring.
  36. 36. These problems were settled with Mendelsdiscovery that the appearance ofcharacters in heredity followed specificlaws.
  37. 37. Mendel showed that the hybrid between two parental types of organisms, which had differed in single character, would produce two types of gametes in equal numbers. Each type of gamete was an unchanged descendant of one of the original parental gametes.
  38. 38.  It soon became apparent from Mendels findings that each hereditary unit (i.e. gene) must be inherited between generations such that each descendant has a physical copy of the material, and that the material must provide information to its carrier as to structure, function and other biological attributes of the organism.
  39. 39.  According to this contention there was no blending or dilution of inheritance whatsoever and inherited characters were determined by discrete units which remained unchanged in the hybrid
  40. 40.  When Mendels findings were discovered forty four years latter they were found to fit quite well with the particulate nature and behaviour of individual chromosomes during cell division. Consequently chromosomes were pinpointed as carriers of hereditary units (genetic material).
  41. 41.  Thence the science of modern genetics was born and proceeded along the line that an actual hereditary material existed, that it was particulate in nature, and that its transmission from one generation to the next could be predicted.
  42. 42. NUCLEIC ACIDS Although the correspondence between the events of cellular division and the transmission of Mendelian characters finally led scientists to identify the nucleus and its constituent chromosomes in particular as carriers of genetic material the nature of the genetic material itself had not been identified by the end of the nineteenth century.
  43. 43.  By this time however, methods of separating nuclei from cytoplasm had been developed. Later an acid containing a large amount of phosphorus was extracted from the nucleus.
  44. 44.  The acid, named nucleic acid, proved to be a constant feature of all cells. Soon it was shown that the acid could be broken down into smaller sections or units each of which consisted a sugar, a phosphate group, and a nitrogen- containing portion. The units were named nucleotides.
  45. 45. It was further shown that the sugar in thenucleotides was either a ribose or a deoxy-ribose.
  46. 46.  No particular nucleic acid contained both these sugars in its molecule. Consequently two main types of nucleic acids could be found.
  47. 47.  Those containing ribose sugar in their molecule were named ribonucleic acids (RNA) while those containing deoxy-ribose sugar were named deoxy-ribonucleic acids (DNA).
  48. 48.  RNA was found to occur mainly in the cytoplasm while DNA occurred only in the nucleus.
  49. 49.  The phosphate group of the nucleotides was shown to be attached to the sugar at its number 5 carbon position. Besides the sugar and phosphate groups which were observed to be constant for all nucleotides of all nucleic acids, a more variable nitrogen-containing group was also present, attached to the sugar at its number 1 carbon position.
  50. 50.  The nitrogen-containing group contained either one or two carbon-nitrogen rings and could function as a base. Bases containing one carbon-nitrogen ring were named pyrimidines, while those containing two rings were named purines.
  51. 51.  Two forms of purines (i.e. adenine and guanine) were identified in both DNA and RNA. Likewise two types of pyrimidines were distinguished (i.e. cytocine and thymine) in DNA while in RNA cytocine was substituted by uracil. Each base distinguished the particular nucleotide carrying it.
  52. 52.  From these observations therefore, it was established that that nucleic acids are linear polymers composed of four types of nucleotides. In DNA the nucleotides are adenine, guanine, cytosine and thymine, whereas in RNA the nucleotides are adenine, guanine, cytosine and uracil.
  53. 53.  Therefore four different types of nucleotides were shown to occur in DNA and in RNA. It was latter shown that the nucleotides were connected together to form chains by a series of phosphate-sugar bonds at number 3 and 5 carbons of the sugar.
  54. 54.  By mid-twentieth century the DNA molecule had been shown to consist of a long chain of hundreds or thousands of different nucleotides in varied lengths and sequences.
  55. 55.  The length and variability of the DNA molecule was consistent with the concept that genetic material could contain different types of information specifying all the biological characteristics of an organism i.e. both the sequence and length of the nucleotide chain (DNA or RNA) could attain a great degree of variability, each sequence and length coding for a different biological message.
  56. 56.  Later in the nineteenth forties x-ray and other studies indicated that the DNA molecule was a double strand.
  57. 57.  The bases of the double-stranded DNA were shown to be quantitatively related such that the total pyrimidine bases (thymine and cytocine) were equal to the total purine bases (adenine and guamine) i.e. T + C = A + G, suggesting that base pairing was always between a pyrimidine and a purine and never between a pyrimidine and pyrimidine nor between a purine and another purine.
  58. 58.  It was established further that each base pair consists of a combination of one purine and one pyrimidine connected together by hydrogen bonds.
  59. 59.  Further it was shown that the pyrimidine thymine was always paired to the purine adenine (i.e. T-A) and the pyrimidine cytocine was bonded to the purine guanine (i.e. C-G).
  60. 60.  It was then confirmed that the DNA molecule is a two-stranded structure and coiled like a rope whose strands could be separated only if the free ends were permitted to revolve freely.
  61. 61. The coiling resembles a which staircasehas been twisted in opposite ways at theend, with the strands composed ofphosphate-sugar linkages which arerepeated continuously, and interconnectedby complementary single purine orpyrimidine bases.
  62. 62.  The great variability possible in the DNA molecule and the ability of the molecule to duplicate itself exactly suggested that the DNA molecule was the very long sought genetic material. Chemical analyses of chromosomes showed that they contained large amounts of DNA together with a histone-type protein, which are now believed to surround DNA. The chromosomes have been shown to contain also small amounts of RNA.
  63. 63.  Further quantitative results showed that the amount of DNA in the nuclei of diploid cells of an organism or species was relatively constant, and that the amount was half as much in haploid cells e.g. sperms.
  64. 64.  This corresponded to the relationship between the diploid and haploid number of chromosomes in cells. This suggested that further that the DNA was the genetic material sought for.
  65. 65.  Cyto-photometric studies with cell nuclei also showed that the DNA remained quantitatively constant in all nuclei of an organism, except in haploid cells in which the amount was only half that present in diploid cells.
  66. 66.  The cyto-photometric technique involves treating the nucleus with a warm acid, followed by staining with a schiffs reagent. This gives a reddish-purplish staining reaction to the nucleus and is very specific to DNA. Thus only chromosomes show this reaction. By measuring the amount of light transmitted through the stained nuclei it is possible to determine quantitatively the amount of DNA in the nuclei.
  67. 67.  It was shown further that the amount of DNA doubled during the interphase stage and was then equally distributed to two daughter cells at anaphase. This again corresponded with the behaviour of chromosomes.
  68. 68.  With the above configuration it was possible to explain how a DNA could replicate itself exactly.
  69. 69.  It was explained that in order to replicate the DNA molecule had to separate its strands and then attract free-floating nucleotides to pair with the bases of each strand. The nucleotides would then be connected into new strands with the aid of enzymes.
  70. 70. Thus it was concluded that the primer DNAstrand enters into a pairing relationshipwith added free nucleotides which are thenzipped together by a polymerase enzyme.
  71. 71.  Further studies showed that new nucleotides in a nucleotide chain were added at number 3 carbon position of the de-oxyribose sugar at one end of the chain. It was observed that if one of the deoxy- ribonucleotides was absent in the reactants then no DNA was synthesized. This suggested that the copying of a DNA strand does not permit the existence of gaps and that this copying proceeds through complementary base pairing.
  72. 72.  The DNA molecule itself was synthesized in vitro for the first time in 1957. This was done by mixing a previously formed DNA with four different kinds of deoxy- ribonucleotides in the form of triphosphates in the presence of a polymerase enzyme and magnesium ions.
  73. 73. REPLICATION AND SYNTHES OF NUCLEIC ACIDS The most accepted explanation of the mode of DNA replication is that each DNA strand is a template or mould for its complement, and a new helix has one old and one newly synthesized strand. It has been suggested that replication starts long before the unwinding of the two complementary DNA strands is completed. The two processes seem to proceed simultaneously.
  74. 74.  Latter it was shown that a DNA molecule can be synthesized in the absence of primer DNA provided the different kinds of nucleotides are mixed with polymerase enzyme. The reaction, though has a long time lag to occur. Eversince different DNA molecules that do not occur naturally have been synthesized in vitro.
  75. 75.  The RNA differs from DNA molecule first because of its ribose sugar and also because of the substitution of the pyrimidine thymine by uracil in RNA. RNA is found throughout the cell
  76. 76.  Three main types of cellular RNA have been identified, i.e. nuclear, ribosomal, and soluble RNA. The latter two RNAs are found in the cytoplasm. Each portion of RNA has been found to represent a special function in the synthesis of proteins.
  77. 77.  RNA appears to maintain a single strand structure. Experimental evidence has shown that, with very few exceptions from some RNA viruses, all cellular RNA is of nuclear origin and is derived from DNA templates. The single strand then separates from the DNA is derived from DNA templates
  78. 78.  Experimental evidence has shown that RNA is copied from only one strand of DNA. The DNA molecule splits and a single strand of the molecule serves as a template upon which a single strand of RNA is assembled.
  79. 79.  The free ends of the bases in the RNA strand become connected in a new ribose- phosphate-base sequence. The single strand then separates from the DNA template and passes into the cytoplasm.
  80. 80.  In RNA the absence of equality between the base ratios of guanine to cytosine and adenine to uracil implies a lack of complementary pairing between the bases. This suggests that RNA generally maintains a single-stranded structure. i.e. there is no double-helix.
  81. 81.  Nevertheless RNA acquires stability from its ability to fold back on itself, so that occasional base pairing and hydrogen bonding enables some form of paired helical structure.