Human Chromosomes and Karyotyping PDF

Human Chromosomes and Karyotyping cabrown Learning outcomes  Describe the structure of chromosomes  Explain the function and importance of telomeres  Describe how chromosomes are packaged  Describe chromosome morphology 2 Human chromosomes...

Human Chromosomes and Karyotyping cabrown Learning outcomes  Describe the structure of chromosomes  Explain the function and importance of telomeres  Describe how chromosomes are packaged  Describe chromosome morphology 2 Human chromosomes and karyotyping Chromosomes  Chromosomes are highly condensed rods of DNA  DNA stores important information about the structure of an animal or plant, and it helps direct the organism as it grows and manages daily tasks.  Chromosomes serve as the storage for this important material, periodically dividing along with cells and replicating to make copies of the DNA they contain.  Chromosomes are also very important in sexual reproduction, as they allow an organism to pass genetic material on to descendants. 3 Human chromosomes and karyotyping  In organisms with cell nuclei, eukaryotes, chromosomes are found inside the nucleus.  Most of these organisms have a set of chromosomes which come in pairs.  In structural cells, each cell retains a complete set of chromosomes, in what is known as diploid form, referring to the fact that the chromosome set is complete.  In cells for sexual reproduction like eggs or sperm, each cell only has half of the parent organism's genetic material, stored in haploid form, ensuring that the parent passes down half of its genes. 4 Human chromosomes and karyotyping  Each pair of chromosomes stores distinct information, and any damage to a chromosome can cause serious problems for the parent organism.  Errors usually occur during cell division, creating gaps in the genetic material in the chromosome.  The morphology of chromosomes is characterized by length, arm ratio, appendages and banding pattern and is usually characteristic for a species. 5 Human chromosomes and karyotyping Basic nomenclature  Diploid- A cell or an organism consisting of two sets of chromosomes: usually, one set from the mother and another set from the father. In a diploid state the haploid number is doubled, thus, this condition is also known as 2n.  Haploid-  (1) The number of chromosomes in a gamete of an organism, symbolized by n.  (2) A cell or an organism having half of the number of chromosomes in somatic cells.  Locus: the site on a chromosome where a particular gene is located 6 Human chromosomes and karyotyping  Size varies from 1 to 30 micron in length and diameter from 0.2 to2 micron. Physical CENTROMERE:- The non-stainable part of the chromosome making a structure primary constriction. CHROMATIDS:- Two chromatids join at the centromere to form a chromosome. CHROMONEMA:- In each chromatid, there are two longitudinal chromonemata coiled with each other. CHROMOMERES:- In each chromonemata, there are “bead” like chromomeres present through out the coil. GENES:- Each chromomeres contains genes, the unit of inheritance of character. SATELLITE:- In some chromosomes a round and elongated satellite is present. CONSTRICTION:- Presence of centromere shows the primary constriction. But in some cases there is an additional Secondary Constriction. 7 Human chromosomes and karyotyping 8 Human chromosomes and karyotyping Chromosome structure  Each end of a chromosome is capped by a telomere, a string of repetitive DNA which protects the chromosome from damage.  Scientists sometimes look at telomeres to gain important information about an organism, as they appear to change over time and they may be associated with aging.  When chromosomes divide to make copies, the telomere also ensures that all of the important genetic material is copied. 9 Human chromosomes and karyotyping  Telomeres function in:  Maintaining structural integrity by preventing the fusion of chromosome ends or protect the ends of chromosomes from degradation by binding to specific telomere-binding proteins.  Ensure complete DNA replication at the tips of chromosmes.  Chromosome positioning. 10 Human chromosomes and karyotyping Chromosome structure - summary 11 Human chromosomes and karyotyping Chemical structure  The major chemical components of the chromosome are nucleic acids (DNA and RNA ), and proteins (histones and nonhistones).  There is, in addition, calcium, which seems to be associated with the DNA  Calcium is important in binding sections of the chromosome together  DNA, RNA and protein all contribute to the chromosomal mass. 12 Human chromosomes and karyotyping  Nucleic acids:  These constitute mostly DNA being about 35% along with histone protein (These are basic proteins having amino acids such as arginine and lysine in their molecules.) which is 55%, thus forming deoxyribonucleoprotein comprising 90% of chromosome.  The remaining 10% part is called residual chromosome and contains RNA 12 to 14%, DNA 2 to 3%, and residual protein 83 to 86%.  The residual protein (nonhistone proteins) is acidic in nature characterized by the preponderance of amino acids tryptophan and tyrosine. The important nonhistone proteins of chromosomes are phosphoproteins, DNA polymerase, RNA polymerase, DPN pyrophosphorylase and nucleoside triphosphatases. 13 Human chromosomes and karyotyping Packaging of DNA into Chromosomes Challenges of Packaging DNA  How to get 2 meters of DNA into nucleus of 6 um  Packaging accomplished w/ help of proteins  Must be compacted in manner that still allows for it to be accessed by enzymes that govern replication, transcription, and repair  Chromosomal Compaction and Histones  A eukaryotic chromosome is a double helix of DNA.  A cell nucleus contains 4 cm of double helix.  This DNA must be compacted, both to fit into the cell nucleus and for accurate segregation in mitosis.  There is an 8000-fold compaction of an extended DNA double helix to make a metaphase chromosome  Winding of DNA on to histones is the first step.  Approximately 160–180 bp of DNA is wrapped around a set of 8 histone proteins (2 each of H2a, H2b, H3 and H4) to form a nucleosome 15 Human chromosomes and karyotyping  Characteristics of Histone Proteins  60 million/cell  Histones are the most abundant proteins in cells.  Histone mass of chromosome = DNA mass  Responsible for packaging DNA into nucleosomes  There are five histones: H1, H2a, H2b, H3, and H4.  Highly conserved, 102-135 aa  Rich in lys and arg  Long N-terminal tail subjected to covalent modification  Specialized variant histones DNA wrapped around eight histone proteins (2 each of histone 2A, 2B, 3, and 4) forms a nucleosome Human chromosomes and karyotyping 17  Nucleosomes can be seen by electron microscopy as 100-Å beadlike structures that are separated by short strands of free double helix.  DNA wrapped around histones forms a “bead-on-a-string” arrangement that comprises the 10-nm or 10-micron fibre.  The 10-micron fibre is further coiled around histone H1 into a thicker and shorter 30-nm or 30-micron fibre. 18 Human chromosomes and karyotyping 19 Human chromosomes and karyotyping  Proteins:  The basic proteins, histone and protamine, the later one is less complex.  Protamine has been found only in spermatozoa of some fishes where it appears to have replaced histone.  Histone is found in practically all the types of nuclei that have been studied.  Histones are highly viscous complex of DNA and may be removed from the chromosome 20 Human chromosomes and karyotyping Model of chromosome organization 21 Human chromosomes and karyotyping  Anatomy of chromosomes There are four important parts in metaphase chromosomes (telomeres, centromeres, and heterochromatin and euchromatin) Human chromosomes and karyotyping 22 Heterochromatin Different types of condensed chromatin with distinct features and roles Found in variety of organisms Represents 10% of chromosome Highly organized, resistant to gene expression Responsible for function of telomeres, centromeres, and may protect genome from transposable elements Euchromatin Less condensed E Composed of all types of chromatin H structures- 30 nm fibers, loops, etc 90% of chromatin Transcribable Human chromosomes and karyotyping 23 Human chromosomes and karyotyping 24 Chromosome Morphology Mitotic chromosomes have been distinguished historically by their relative size and centromere placement. The centromere is the site of attachment of the chromosome to the spindle apparatus. – The connection is made between microtubules of the spindle and a protein complex, called the kinetochore, that assembles at the centromere sequences Human chromosomes and karyotyping 25  Chromosomes are metacentric, submetacentric, acrocentric, or telocentric, depending on the placement of the centromere The placement of the centromere divides the chromosome into arms. Human chromosomes and karyotyping 26  There are no telocentric human chromosomes. Human chromosomes are acrocentric Short or submetacentric and so have long and arm (p) short arms. The long arm of a chromosome is designated q, and the short arm is designated p. Long Acrocentric chromosomes have a long arm arm (q) length:short arm length ratio of from 3:1 to 10:1. Chromosomes 13 to 15, 21, and 22 are acrocentric. Human chromosomes and karyotyping 27 Classification of Chromosomes by Size and Centromere Position Human chromosomes and karyotyping 28  Chromosome Number The number and appearance of chromosomes is an important characteristic in genetic analyses. The number of chromosomes per organism is always a definite number Human chromosomes and karyotyping 29  Visualizing Chromosomes Conventional cytological stains, such as Feulgen’s, Wright’s, and hematoxylin, have been used to visualize chromosomes. An advance in the recognition of individual chromosomes was the demonstration that fluorescent stains and chemical dyes can react with specific chromosome regions. This region-specific staining results in the formation of band patterns where portions of the chromosome accept or reject the stain Human chromosomes and karyotyping 30  When chromosomes are stained with the fluorescent dyes quinacrine and quinacrine mustard, the resulting fluorescence pattern visualized after staining is called Q banding Each human chromosome can be identified by its characteristic banding pattern. Q banding gives a particularly intense staining of the human Y chromosome and thus may also be used to distinguish the Y chromosome in interphase nuclei. Q banding requires a fluorescent microscope The chemical dye Giemsa stains in patterns, or G bands, similar to those seen in Q banding Human chromosomes and karyotyping 31 Q bands G-bands Human chromosomes and karyotyping 32  Harsher treatment of chromosomes (87oC for 10 minutes, then cooling to 70oC) before Giemsa staining will produce a pattern opposite to the G banding pattern called R banding. R bands can also be visualized after staining with acridine orange. Alkali treatment of chromosomes results in centromere staining, or C banding. Centromere staining is absent in G band patterns. Human chromosomes and karyotyping 33 C-banded human male metaphase spread Human chromosomes and karyotyping 34  Nucleolar organizing region staining (NOR staining) is another region-specific staining approach. Chromosomes treated with silver nitrate will stain specifically at the constricted regions, or stalks, on the acrocentric chromosomes. Staining of chromosomes with 4′,6-diamidino-2-phenylindole (DAPI) is a way to detect mycoplasmal contamination in cell cultures. DAPI binds to the surface grooves of double-stranded DNA and fluoresces blue under ultraviolet light (353-nm wavelength). DAPI can be used to visualize chromosomes as well as whole nuclei. Human chromosomes and karyotyping 35  Chromosome banding facilitates detection of small deletions, insertions, inversions, and other abnormalities and the identification of distinct chromosomal locations For this purpose, the reproducible G-banding pattern has been ordered into regions, comprising bands and sub-bands. For example, in a site on the long arm (q) of chromosome 17 is located in region 1, band 1, sub-band 2, or 17q11.2 Human chromosomes and karyotyping 36  Defining Chromosomal Location Arm Region Band Subband Identification of 3 2 2 1 2 chromosomal location by 2 p 1 1 5 1 G-band patterns. 4 1 3 2 Locations are designated 1 1 17 q11.2 by the chromosome 1 1 2 3 number 17 in this q 2 1 2 3 example, the arm q, the 3 1 2, 3 4 region 1, the band 2 4 1 2 Chromosome 17 3 1 and the sub-band 2 Human chromosomes and karyotyping 37  Chromosomes versus Genes Chromosome: made of many genes Gene: DNA sequence that codes for a trait Allele: alternate form of a gene Examples: eye color gene has blue, brown, green alleles Human chromosomes and karyotyping 38  Karyotype Karyotyping reveals characteristics of an individual’s chromosomes Karyotype – Image of an individual’s complement of chromosomes arranged by size, length, shape, and centromere location – describes the amount of chromosome count and morphology of an organism under the light microscope. Human chromosomes and karyotyping 39 The steps in the Add a few Add phytohemagglutinin to drops of blood. stimulate mitosis. process of creating a karyotype for Draw 10 to 20 ml chromosome analysis of blood. Incubate at 37°C for 2 to 3 days. Transfer to tube Transfer cells Add Colcemid to culture containing fixative. to tube. for 1 to 2 hours to stop mitosis in metaphase. Centrifuge to concentrate cells. Add low-salt solution to eliminate red blood cells and swell lymphocytes. Drop cells onto microscope slide. Examine with Digitized microscope. chromosome images Stain slide processed to make with Giemsa. karyotype. Human chromosomes and karyotyping 40 Constructing a Karyotype Human chromosomes and karyotyping 41  Human Chromosomes: Humans have 23 pairs of chromosomes – 22 pairs are called autosomes (they are the same in males and females – Our genetic information is stored in 23 pairs of chromosomes that vary widely in size and shape. – Chromosome 1 is the largest and is over three times bigger than chromosome 22. Human chromosomes and karyotyping 42 Human chromosomes and karyotyping 43 Sex Chromosomes One pair of chromosomes differ in males and females, – Called sex chromosomes, there are two types: X chromosome (similar to all other chromosomes) Y chromosome (shorter and hook shape) Human chromosomes and karyotyping 44  Sex Chromosomes Females: Have two X chromosomes Males: Have one x chromosomes (one from mother and one from (from mother) and one y (from father) father) Human chromosomes and karyotyping 45 The human X (left) and Y chromosomes, magnified about 10,000 times Human chromosomes and karyotyping 46 Sex Chromosomes – More than 1400 genes are found on the X chromosome, some of which are shown. – The human Y chromosome is much smaller than the X chromosome and contains only about 200 genes, most of which are associated with male sex determination and sperm development. Human chromosomes and karyotyping 47 Human chromosomes and karyotyping 48