Chromosome structure. Classification of human chromosome. Telomere, telomeric DNA sequences. Structural features of a typical human gene.

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1. Lecture#4 Chromosome structure. Classification of human chromosome. Telomere, telomeric DNA sequences. Structural features of a typical human gene. Lecturer Elene Petriashvili

2. Genetic continuity between generations of cells and between generations of sexually reproducing organisms is maintained through the processes of mitosis and meiosis, respectively.

3. Diploid eukaryotic cells contain their genetic information in pairs of homologous chromosomes, with one member of each pair being derived from the maternal parent and one from the paternal parent.

4. The medical relevance of these processes lies in errors of one or the other mechanism of cell division, leading to the formation of an individual or of a cell lineage with an abnormal number of chromosomes and thus an abnormal dosage of genomic material.

5. Meiotic nondisjunction, particularly in oogenesis, is the most common mutational mechanism in our species, responsible for chromosomally abnormal fetuses in at least several percent of all recognized pregnancies.

6. Among pregnancies that survive to term, chromosome abnormalities are a leading cause of developmental defects, failure to thrive in the newborn period, and intellectual disability.

7. Mitotic nondisjunction in somatic cells also contributes to genetic disease. Nondisjunction soon after fertilization, either in the developing embryo or in extraembryonic tissues like the placenta, leads to chromosomal mosaicism that can underlie some medical conditions, such as a proportion of patients with Down syndrome.

8. Further, abnormal chromosome segregation in rapidly dividing tissues, such as in cells of the colon, is frequently a step in the development of chromosomally abnormal tumors, and thus evaluation of chromosome and genome balance is an important diagnostic and prognostic test in many cancers.

9. Chromosomes , found in the cell nucleus, contain many genes. A gene is a section of DNA, which carries coding for a particular protein.

10. Different genes control the development of different characteristics of an organism. Many genes are needed to carry all the genetic information for a whole organism.

11. Homologous chromosomes have important genetic similarities: containing identical gene sites along their lengths called a locus (pl. loci); they are identical in the traits influence and in their genetic potential. In a population of members of the same species, many different alternative forms of the same gene, called alleles, can exist.

12. Two specific DNA structures are essential for the maintenance of a constant chromosome complement in a given species: centromeres and telomeres. Centromeres consist of DNA sequences that, together with 90 or more proteins, direct the segregation of chromosomes during cell division.

13. Telomeres are specialized structures that protect the ends of chromosomes and permit complete replication of the chromosomal DNA. The first levels of packaging shorten the DNA about 40-fold by wrapping it around histone proteins to form nucleosomes.

14. The complex of DNA with its packaging proteins is called chromatin. Nuclei contain two broad classes of chromatin: heterochromatin, which is highly condensed throughout the cell cycle and is generally inactive in transcription, and euchromatin , which is less condensed and contains actively transcribed genes.

15. Chromatin is found in two varieties:  euchromatin  and  heterochromatin . Originally, the two forms were distinguished cytologically by how intensely they stained – the euchromatin  is less intense, while  heterochromatin  stains intensely, indicating tighter packing.

16. Euchromatin contains less DNA while  heterochromatin   contains more DNA.  Euchromatin  is early replicative while  heterochromatin  is late replicative.   Euchromatin  is found in eukaryotes, cells with nuclei, and prokaryotes, cells without nuclei.

17. Euchromatin  is a lightly packed form of chromatin (DNA, RNA, and protein) that is enriched in genes, and is often (but not always) under active transcription. Euchromatin  comprises the most active portion of the genome within the cell nucleus. 92% of the human genome is  euchromatic .

18. Different types of chromatin are defined by complex patterns of posttranslational modifications of the histone proteins. These modifications direct the binding of protein readers that establish chromatin states to promote or repress gene expression or serve other structural roles.

19. The Every organism is defined by a blueprint consisting of information stored in its chromosomes. With the exception of a few viruses, these chromosomes are composed of enormously long circular or linear molecules of DNA.

20. Chromosomes have fascinated biologists ever since it was understood that they contain the genetic information that defines each organism—its genome. After Watson and Crick’s proposal of a structure for DNA in 1953, it was realized that the DNA is a linear sequence of A, T, G, and C bases that can be thought of as a code to describe the physical attributes for every organism.

21. The Every organism is defined by a blueprint consisting of information stored in its chromosomes. With the exception of a few viruses, these chromosomes are composed of enormously long circular or linear molecules of DNA.

22. In prokaryotes, the single chromosome is concentrated in a specialized region of the cytoplasm called the nucleoid. In eukaryotes, the chromosomes are packaged in a specialized membrane-bounded compartment known as the nucleus. This difference in organization has important consequences for the regulation of gene expression.

23. Chromosomes are enormous DNA molecules that can be propagated stably through countless generations of dividing cells (Fig. 7.1). Genes are the reason for the existence of the chromosomes, but in higher eukaryotes, they make up a relatively small fraction of the chromosomal DNA.

24. Cells package chromosomal DNA with roughly twice its weight of protein. This DNA-protein complex, called chromatin. In addition to the genes, only three classes of specialized DNA sequences are needed to make a fully functional chromosome: a centromere, (b) two telomeres, and (c) an origin of DNA replication for approximately every 100,000 base pairs ( bp ).

25. Centromeres regulate the partitioning of chromosomes during mitosis and meiosis. Telomeres protect the ends of the chromosomal DNA molecules and ensure their complete replication.

26. There are 24 different types of human chromosome, each of which can be distinguished cytologically by a combination of overall length, location of the centromere, and sequence content, the latter reflected by various staining methods. The centromere is apparent as a primary constriction, a narrowing or pinching-in of the sister chromatids due to formation of the kinetochore.

27. This is a recognizable cytogenetic landmark, dividing the chromosome into two arms, a short arm designated p (for petit ) and a long arm designated q .

28. Depending on the position of the centromere, different arm ratios are produced. The shorter arm, by convention, is shown above the centromere and is called the p arm (p, for “petite”). The longer arm is shown below the centromere and is called the q arm (q because it is the next letter in the alphabet).

29. The ends of each chromosome (or chromatid) are marked by telomeres , which consist of specialized repetitive DNA sequences that ensure the integrity of the chromosome during cell division. Correct maintenance of the ends of chromosomes requires a special enzyme called telomerase, which ensures that the every ends of each chromosome are replicated.

30. Human chromosomes are often classified into three types that can be easily distinguished at metaphase by the position of the centromere, the primary constriction visible at metaphase; metacentric chromosomes, with a more or less central centromere and arms of approximately equal length; submetacentric chromosomes, with an off-center centromere and arms of clearly different lengths; and acrocentric chromosomes, with the centromere near one end.

31. A potential fourth type of chromosome, telocentric, with the centromere at one end and only a single arm, does not occur in the normal human karyotype, but it is occasionally observed in chromosome rearrangements.

32. Metacentric Chromosomes Metacentric chromosomes have the centromere in the center, such that both sections are of equal length. Human chromosome 1 and 3 are metacentric

33. Submetacentric Chromosomes Submetacentric chromosomes have the centromere slightly offset from the center leading to a slight asymmetry in the length of the two sections. Human chromosomes 4 through 12 are submetacentric .

34. Acrocentric Chromosomes Acrocentric chromosomes have a centromere which is severely offset from the center leading to one very long and one very short section. Human chromosomes 13, 15, 21, and 22 are acrocentric .

35. Polytene Chromosomes Giant polytene chromosomes are found in various tissues (salivary, midgut , rectal, and malpighian excretory tubules) in the larvae of some flies, as well as in several species of protozoans and plants. They were first observed by E. G. Balbiani in 1881.

36. The large amount of information obtained from studies of these genetic structures provided a model system for subsequent investigations of chromosomes. Each polytene chromosome is 200 to 600 mm long, and when they are observed under the light microscope, they exhibit a linear series of alternating bands and interbands .

37. Lampbrush Chromosomes The lampbrush chromosome, so named because it resembles the brushes used to clean kerosene lamp chimneys in the nineteenth century. First discovered in 1882 by Walther Flemming in salamander oocytes, and then seen in 1892 by J. Ruckert in shark oocytes. They are now known to be characteristic of most vertebrate oocytes, as well as the spermatocytes of some insects.

38. Therefore, they are meiotic chromosomes. Most of the experimental work on them has been done with material taken from amphibian oocytes. These unique chromosomes are easily isolated from oocytes in the diplotene stage of the first prophase of meiosis, where they are active in directing the metabolic activities of the developing cell.

39. The homologs are seen as synapsed pairs held together by chiasmata. Lampbrush chromosomes are often extended to lengths of 500 to 800 mm. Lampbrush chromosomes are interpreted as being extended, uncoiled versions of the normal meiotic chromosomes.

40. Key Terms Centromere: The chromosomal locus that regulates the movements of chromosomes during mitosis and meiosis. The centromere is defined by specific DNA sequences plus proteins that bind to them, although epigenetic factors also play a key role. In higher eukaryotes, the centromere of mitotic chromosomes can be visualized as a constricted region where sister chromatids are held together most closely.

41. Chromatin: DNA plus the proteins that package it within the cell nucleus. Chromosome: A DNA molecule with its attendant proteins that moves as an independent unit during mitosis and meiosis.

42. Before DNA replication, each chromosome consists of a single DNA molecule plus proteins and is called a chromatid. After replication, each chromosome consists of two identical DNA molecules plus proteins. These are called sister chromatids. Chromosomal DNA molecules are usually linear but can be circular in organelles, bacteria, and viruses.

43. Kinetochore: The centromeric substructure that binds microtubules and directs the movements of chromosomes in mitosis. Telomere: The specialized structure at either end of the chromosomal DNA molecule that ensures the complete replication of the chromosomal ends and protects the ends within the cell.

44. The essential nature of these structural elements of chromosomes and their role in ensuring genome integrity is illustrated by a range of clinical conditions that result from defects in elements of the telomere or kinetochore or cell cycle machinery or from inaccurate replication of even small portions of the genome.

45. Single copies of unique DNA sequences that make up genes, a great deal of the DNA sequencing within eukaryotic chromosomes is repetitive in nature and that various levels of repetition occur within the genomes of organisms. Figure 12–14 schematizes these categories.

46. Satellite DNA - nucleotide composition (e.g., the percentage of G‚C versus A“T pairs) of the DNA of a particular species is reflected in the DNA’s density, which can be measured with sedimentation equilibrium centrifugation. Satellite DNA, makes up a variable proportion of the total DNA, depending on the species.

47. Satellite DNA is found in the heterochromatic centromeric regions of chromosomes.

48. What are the differences between satellite DNA and repetitive DNA? Repetitive DNA is the part of the DNA which has repetitin of the base pairs no matter how much and in what way like palindrome repeats or mirror repeats or trinucleotide repeats or simple repeats. Satellite DNA is the part of the repetitive DNA which is very specific in nature; DNA that contains many tandem (not inverted) repeats of a short basic repeating unit.

49. Satellite DNA is located at very specific spots ( a particular place or point) in the genome (on chromosomes 1, 9, 16 and the Y chromosome, the tiny short arms of chromosomes 13-15 and 21 and 22, and near the centromeres of chromosomes).

50. The degree of repetition is on the order of 1000 to 10 million at each locus. Loci are few, usually one or two per chromosome. They were called satellites since in density gradients, they often sediment as distinct, satellite bands separate from the bulk of genomic DNA owing to a distinct base composition. Satellite DNA repeats which are about 25 nucleotides is called microsatellite sequences.

51. There are different kinds of repeats. The 3 major types include Terminal Repeats Tandem Repeats Interspersed repeats

52. Satellite DNA are a sub type of tandem repeats along with minisatellites and microsatellites.  Microsatellites - repeat units less than 10 base pairs in length present in terminal ends of chromosomes. Minisatellites - much longer in length. Usually 10-60 base pairs. They can be present in any part of the genome including centromere.

53. Telomeric DNA Sequences The telomere—the structure that “caps” the ends of linear eukaryotic chromosomes. The heterochromatic cap structure renders chromosome ends inert in interactions with other chromosome ends and with enzymes that use double-stranded DNA ends as substrates (such as repair enzymes).

54. Telomeric DNA sequences consist of short tandem repeats. It is this group of repetitive sequences that contributes to the stability and integrity of the chromosome. The number of copies making up a telomere varies in different organisms, and there may be as many as 1000 repeats in some species.

55. A central question concerns the role of these repeat DNA sequences in telomere function. Recent findings have established that the sequences are transcribed and that the RNA product, called TERRA (telomere repeat-containing RNA), is an integral component of the telomere, contributing to its heterochromatic nature by facilitating methylation of the histone H3K9.

56. TERRA (telomere repeat-containing RNA sequences) are complementary to those of the RNA component of telomerase, which provides the template for the synthesis of telomeric DNA. Serving as a telomerase ligand, TERRA acts as an inhibitor of telomerase.

57. Telomerase is active in germ-line cells but is inactive in somatic cells. And, in human cancer cells, which have become immortalized, the transition to malignancy appears to require the activation of telomerase in order to overcome the normal senescence associated with chromosome shortening.

58. Nuclear Genes It is estimated that there are between 25,000 and 30,000 genes in the nuclear genome. The distribution of these genes varies greatly between chromosomal regions. For example, heterochromatic and centromeric regions are mostly non-coding, with the highest gene density observed in subtelomeric regions.

59. Chromosomes 19 and 22 are gene rich, whereas 4 and 18 are relatively gene poor. The size of genes also shows great variability: from small genes with single exons to genes with up to 79 exons .

60. Gene Structure The original concept of a gene as a continuous sequence of DNA coding for a protein was turned on its head in the early 1980s by detailed analysis of the structure of the human β-globin gene.

61. It was revealed that the gene was much longer than necessary to code for the β-globin protein, containing non-coding intervening sequences, or introns, that separate the coding sequences or exons. Most human genes contain introns, but the number and size of both introns and exons is extremely variable.

62. A gene can be visualized as a segment of a DNA molecule containing the code for the amino acid sequence of a polypeptide chain and the regulatory sequences necessary for its expression. The majority of genes are interrupted by one or more noncoding regions. These intervening sequences, called introns, are initially transcribed into RNA in the nucleus but are not present in the mature mRNA in the cytoplasm.

63. Introns alternate with exons, the segments of genes that ultimately determine the amino acid sequence of the protein, as well as certain flanking sequences that contain the 5′ and 3′ untranslated regions (Fig. 3-4).

64. Eukaryotic gene structure: Most eukaryotic genes in contrast to typical bacterial genes, the coding sequences (exons) are interrupted by noncoding DNA (introns). The gene must have (Exon; start signals; stop signals; regulatory control elements).

65. Although a few genes in the human genome have no introns, most genes contain at least one and usually several introns. Surprisingly, in many genes, the cumulative length of the introns makes up a far greater proportion of a gene’s total length than do the exons.

66. Pseudogenes Particularly fascinating is the occurrence of genes that closely resemble known structural genes but which, in general, are not functionally expressed: so-called pseudogenes.

67. These are thought to have arisen in two main ways: either by genes undergoing duplication events that are rendered silent through the acquisition of mutations in coding or regulatory elements, or as the result of the insertion of complementary DNA sequences, produced by the action of the enzyme reverse transcriptase on a naturally occurring messenger RNA transcript, that lack the promoter sequences necessary for expressio n.

68. Tandemly repeated DNA sequences consist of blocks of tandem repeats of non-coding DNA that can be either highly dispersed or restricted in their location in the genome. Tandemly repeated DNA sequences can be divided into three subgroups: satellite, minisatellite , and microsatellite DNA.

69. Satellite DNA Satellite DNA accounts for approximately 10% to 15% of the repetitive DNA sequences of the human genome and consists of very large series of simple or moderately complex, short, tandemly repeated DNA sequences that are transcriptionally inactive and are clustered around the centromeres of certain chromosomes.

70. The telomeric repeat sequences are necessary for chromosomal integrity in replication and are added to the chromosome by an enzyme known as telomerase. Hypervariable minisatellite DNA is made up of highly polymorphic DNA sequences consisting of short tandem repeats of a common core sequence.

71. What is the genetic code? The genetic code = the sequence of bases found along the mRNA molecule There are only four letters to this code (A, G, C and U) The code needs to be complex enough to represent 20 different amino acids used to build proteins. The genetic code is read in triplets Each amino acid in a protein is specified by a group of three bases in messenger RNA

72. The genetic code: Is redundant – meaning the most of the 20 amino acids are specified by more than one codon Is unambiguous – a single codon never codes for more than one amino acid It is nearly universal – all codons specify the same amino acids in all organisms It is conservative – when several codons specify the same amino acid, the first two bases are almost always identical

73. Clinical cytogenetics is the study of chromosomes, their structure and their inheritance, as applied to the practice of medical genetics. It has been apparent for nearly 50 years that chromosome abnormalities—microscopically visible changes in the number or structure of chromosomes—could account for a number of clinical conditions that are thus referred to as chromosome disorders.

74. Chromosome analysis—improved resolution and precision at both the cytological and genomic levels—is an increasingly important diagnostic procedure in numerous areas of clinical medicine. Chromosome disorders form a major category of genetic disease. Specific chromosome abnormalities are responsible for hundreds of identifiable syndromes that are collectively more common than all the mendelian single-gene disorders together.

75. INTRODUCTION TO CYTOGENETICS AND GENOME ANALYSIS The general morphology and organization of human chromosomes, as well as their molecular and genomic composition. To be examined by chromosome analysis for clinical purposes, cells must be capable of proliferation in culture.

76. The most accessible cells that meet this requirement are white blood cells, specifically T lymphocytes. To prepare a short-term culture that is suitable for cytogenetic analysis of these cells, a sample of peripheral blood is obtained, and the white blood cells are collected, placed in tissue culture medium, and stimulated to divide.

77. After a few days, the dividing cells are arrested in metaphase with chemicals that inhibit the mitotic spindle. Cells are treated with a hypotonic solution to release the chromosomes, which are then fixed, spread on slides, and stained by one of several techniques, depending on the particular diagnostic procedure being performed. They are then ready for analysis. Although ideal for rapid clinical analysis, cell cultures prepared from peripheral blood have the disadvantage of being short-lived (3 to 4 days).

78. Long-term cultures suitable for permanent storage or further studies can be derived from a variety of other tissues. Skin biopsy, a minor surgical procedure, can provide samples of tissue that in culture produce fibroblasts, which can be used for a variety of biochemical and molecular studies as well as for chromosome and genome analysis.

79. White blood cells can also be transformed in culture to form lymphoblastoid cell lines that are potentially immortal. Bone marrow has the advantage of containing a high proportion of dividing cells, so that little if any culturing is required; however, it can be obtained only by the relatively invasive procedure of marrow biopsy.

80. Its main use is in the diagnosis of suspected hematological malignancies. Fetal cells derived from amniotic fluid ( amniocytes ) or obtained by chorionic villus biopsy can also be cultured successfully for cytogenetic, genomic, biochemical, or molecular analysis.

81. Chorionic villus cells can also be analyzed directly after biopsy, without the need for culturing. Remarkably, small amounts of cell-free fetal DNA are found in the maternal plasma and can be tested by whole-genome sequencing.

82. Molecular analysis of the genome, including whole genome sequencing, can be carried out on any appropriate clinical material, provided that good-quality DNA can be obtained.

83. Cells need not be dividing for this purpose, and thus it is possible to study DNA from tissue and tumor samples, for example, as well as from peripheral blood. Which approach is most appropriate for a particular diagnostic or research purpose is a rapidly evolving area as the resolution, sensitivity, and ease of chromosome and genome analysis increase.

84. Thank you for attention