Veterinary Genetics & Genetic Engineering Notes (Mansoura University) PDF

# Notes on Veterinary Genetics & Genetic Engineering ## Mansoura University Faculty of Veterinary Medicine Department of Husbandry & Department **First Year of General Program** **First Term** **Compiled By** Staff Members of Genetics and Genetic Engineering ## Introduction of Genetics * **Gen...

# Notes on Veterinary Genetics & Genetic Engineering ## Mansoura University Faculty of Veterinary Medicine Department of Husbandry & Department **First Year of General Program** **First Term** **Compiled By** Staff Members of Genetics and Genetic Engineering ## Introduction of Genetics * **Genetics:** The study of heredity and variation * **Hereditary:** How traits pass from one generation to the next. * **Variation:** Accounts for differences between individuals between species. ## The areas (branches) (approaches) (disciplines) of Genetics 1. **Classical Genetics (Mendelian)** * It is also known as "transmission genetics." * Mendel's first and second laws of inheritance 2. **Cytogenetics** * Combined study of cytology includes changes in chromosome number and structure. 3. **Molecular Genetics (biochemical)** * This is the study of the genetic materials (DNA and RNA) 4. **Population Genetics** * This is the study of gene and genotypic frequencies in the population 5. **Radiation genetics** * Deal with effect of radiations on chromosomes and genes. 6. **Microbial genetics** * Deal with inheritance of characters in microorganisms ## Chromosomes and cell cycle * **Definition of chromosome:** In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called chromosomes. It is a carrier of genetic information that is visible under an ordinary **light microscope**. * **Each chromosome** is made up of DNA tightly coiled many times around proteins called **histones** that support its structure. In other words, it is the DNA complexed with protein called histones. * **There is two type of protein** is isolated in the cell: histone protein and non-histone protein. * **Histone proteins:** Such as proteins that DNA is wrapped around them to form chromosome. * **Non-histone protein:** Such as: 1. **Enzymes:** Used in different DNA activities: DNA repair, replication, and translation such as DNA polymerase and DNA ligase. 2. **Scaffold proteins:** Whose main function is to bring other proteins together to interact. They play the role of skeleton. 3. **Other motor proteins:** All play essential roles in cell structure and regulatory functions that make life possible, (the muscle protein myosin which motors the contraction of muscle fibers). * **Histones:** Are basic proteins as they contain a large amount of basic amino acids **arginine and lysine**. They have positive charges due to positive amino acids, that allow them to associate with DNA (negative charge due to phosphate group in the DNA backbone that carry negatively charged oxygen). **There are five types of histones, four main types (core-histones)** H2A, H2B, H3, and H4 which are the same in their structure in most species called histone octamer. They are present 2 each of every 200 base pairs of DNA. * **H1 histone:** Is the fifth type that is present only once every 200 base pairs. These two turns sealed off by one H1 molecule. * **The two turns** contain about 146 base pairs. * **The remaining part of 200 base pair** form linker DNA connecting the nucleosomes. * **The DNA is coiled outside of the octamer** as it makes two complete turns around it. * **Function of histones:** It is involved in maintenance of high order folding of chromatin and to compact DNA so impacting gene regulation. * **Without histones**, the unwound DNA in chromosomes would be very long. * **Chromatin:** Is composed of DNA and proteins (histones) that make up chromosomes which are packaged into thin, stringy fibers called nucleosome. * **The function of chromatin:** Package of DNA into a small volume to fit into the nucleus of a cell and protect the DNA structure. Packaging DNA into chromatin allows for mitosis and meiosis, prevents chromosome breakage and controls gene expression and DNA replication. ## Nucleosome * It is the functional subunit of chromatin. A nucleosome is a section of DNA that is wrapped around a core of proteins (histones) inside the nucleus. * **The function of nucleosomes:** Reduce the overall length of DNA in the nucleus, helping the DNA to be condensed into a smaller volume inside the nucleus of cell. ## Types of Chromatin | Items | Heterochromatin | Euchromatin | | -----------|-----------------------------------|--------------------------------------------------| | Genes | Contain inactive gene | Contain active gene | | Stain | Dark staining regions (condensed) | Light staining regions (less condensed) | | Replication | Contain repetitive DNA sequences and it is late replicating (replicated when the bulk of DNA has already been replicated) and is not transcribed. | It represents most of the chromatin which contains structural genes which replicate and transcribe because DNA is more accessible, contains the most active portion of the genome within the cell nucleus. | | Position | Centromeric regions and telomeric regions | Distal arms of the chromosome | | Function | Gene regulation and protection of chromosome integrity | Participates in the active transcription of DNA to mRNA products. The unfolded structure allows regulatory proteins and RNA polymerase complexes to bind to the DNA sequence, which can then initiate the transcription process. | ## Cell cycle * **The cell cycle** is the series of events that take place in a cell leading to duplication of its DNA and separation of cell into daughter cells. Duration of cell cycle between cells is variable. ## Phases of cell cycle 1. **Interphase (95% of cell cycle period):** * **G1 (growth and metabolic activity):** The cell grows, duplicates organelles, and makes the molecular building blocks it will need in later steps (enzymes and proteins). * **S (synthetic activity):** The cell synthesizes a complete copy of the DNA in its nucleus. * **G2 (growth and metabolic activity):** The cell grows more, makes proteins and organelles, and begins to reorganize its contents in preparation for mitosis. 2. **Division phase (5% of cell cycle period):** * **Mitotic (M) phase**, the cell divides its copied DNA and cytoplasm to make two new cells. M phase involves mitosis (nuclear) and cytokinesis (cytoplasmic) divisions. * **The G0 phase:** Is a period in the cell cycle in which cells exist in a quiescent state. Nerve and heart cells enter G0 phase permanently. Some cells enter G0 temporarily until an external signal activates the onset of G1. * **Function of cell cycle:** Duplicate accurately the amount of DNA in the chromosomes and then segregate the copies into two genetically identical daughter cells. ## Regulation of cell cycle * **Both external and internal factors regulate the cell cycle** in eukaryotic cells. * **External factors:** * External factors that help regulate the cell cycle include physical and chemical signals. One example of a physical signal is cell-to-cell contact. When external factors bind to their receptors, they can activate internal factors that affect the cell cycle. * **There are group of proteins (enzymes) known as Cyclin-dependent kinases (CDKs)** have role in regulating the cell cycle. CDK binds a regulatory protein called **cyclin** to form cyclin-CDK complex making it functional and allowing it to modify target proteins. * **CDK works by phosphorylation of cyclin** through transferring phosphate groups from ATP to some amino acids in the cyclin (attach phosphate groups to specific target proteins). * **Anaphase is activated** by the destruction of the proteins that hold the sister chromatids together (Cohesion). The Metaphase Cdk is then inactivated by cyclin proteolysis, which leads to cytokinesis and the end of Mitosis phase. CDK/cyclin complexes must be kept inactive prior to mitosis in order to ensure appropriate timing of mitotic entry. * **Specific enzymes break down cyclins at defined times** in the cell cycle. When cyclin levels decrease, CDKs become inactive. Cell cycle arrest can occur if cyclins fail to degrade. * **In general, Cdk levels remain relatively constant across the cell cycle**, but Cdk activity and target proteins change as levels of vaious cyclins rise and fall. * **Cancer results from a disruption of the normal regulation of the cell cycle.** When the cycle proceeds without control, cells can divide without order and accumulate genetic defects that can lead to a cancerous tumor. ## Checkpoints of cell cycle * **There are 3 checkpoints within the cell cycle** that indicate points at which cell cycle can be stopped in order to ensure the accuracy of the process: 1. **G1 checkpoint:** The G1 checkpoint control mechanism ensures that everything is ready for DNA synthesis. 2. **G2 checkpoint:** The checkpoint lies between G2 phase and M phase, the DNA is checked along with other features of the cells in order to ensure that it is suitable to undergo mitosis. Ensures new daughter cell receives all the genetic information needed. 3. **Metaphase checkpoint:** During metaphase spindle fibers attach to the centromeres of chromosomes allowing the separation of homologous chromatids in anaphase. The checkpoint is aimed at ensuring the correct attachment of spindle fibers and the presence of molecules that inhibit anaphase until attachment has occurred. ## Mitotic chromosomes * **The chromosomes can be classified in shape according to position of centromere into four types:** 1. **Telocentric:** the centromere located at one end of chromosome. 2. **Acrocentric:** the chromosome has one short arm and one long arm. 3. **Submetacentric:** the chromosome have unequal long arm. 4. **Metacentric:** the chromosome have equal long arm. * **The chromosomes can be classified according to number of centromeres into four types:** 1. **Monocentric:** Most of chromosomes have only one centromere 2. **Holocentric:** Diffuse centromeres along the length of chromosome. 3. **Acentric:** chromosome doesn't have any centromere. So it not bind to spindle fibers and lost during division. 4. **Dicentric:** chromosome has two centromeres. So lost during division as it fragmented due to grasping by spindle fibers in different directions. ## Chromosomal structure * **Each chromosome has two arms**, the p (short) arm and the q (long) arm. 1. **Chromatid:** * During metaphase, each chromosome consists of two symmetrical structures, each of them called chromatid. * **The two sister chromatids connected together by centromere which divided longitudinally during anaphase.** Anaphase chromosome has only one chromatid while metaphase chromosome has two sister chromatid. 2. **Centromere:** * **Definition:** the region of chromosome that attached to spindle fibers. * **It is a disc shape structure lies in a thinner segment of chromosome called primary constriction.** It bind to microtubules of spindle fibers. * **Contain highly repetitive DNA that condensed with protein and stained dark (heterochromatin).** 3. **Telomeres:** * **Definition:** Are caps found at the end of chromosomes * **Function:** Provide stability to the chromosome ends and prevent stickiness of chromosome, contains heterochromatin. 4. **Secondary constriction:** * **Definition:** It is a thin segment of chromosome which constant in its position and extent and not found in all chromosomes. * **Function:** It is useful in identifying certain chromosomes. * It is differentiated from primary constriction by binding to spindle fibers. 5. **Nucleolar organizer region (NOR):** * **Definition:** it is the region which contain the genes coding for 18s and 28s rRNA and that induce the formation of nucleolus. * **Function:** It is found in certain chromosomes. It is useful in identifying certain chromosomes. 6. **Satellite:** * **Definition:** it is a rounded body separated from the rest of chromosome by secondary constriction. * **Function:** It is useful in identifying certain chromosomes. ## Common terms: | | | | |---------------------------|-------------------------|---------------------------| | **Chromatin** | **Chromosome** | **Chromatid** | | Form of genetic material during interphase of cell cycle | Form of genetic material during division phase of cell cycle | Part of chromosome, one arm of chromosome | | Less coiled, less condensed and thin | More coiled, More condensed and thick | Coiled, condensed and thick | | Interphase, early prophase and late telophase | Late prophase, metaphase, anaphase and early telophase | Anaphase and early telophase | ## Evaluation of genomic size | C value | C value paradox (C value enigma) | G value | G value paradox | |--------------------|--------------------------------------------------------------------------------------------------------------------------|-------------------|---------------------------------------------------------------------------------------------------------------| | Is the amount in pictograms (trillionths 10-12 of a gram, abbreviated as pg) of DNA contained within a nucleus of an eukaryotic organism. | Is the amount of DNA in a haploid genome does not seem to correspond to the complexity of an organism | Which represents the number of genes in an organism | Lack of association between the number of genes of an organism and its complexity. | | It correlates inversely with cell division rate, meaning that large genomes are typically found in the nuclei of large, slowly dividing cells. | For example: Mammals have 30,000 to 50,000 genes, but their genome size is 3 x 10⁹ bp. | | The microscopic nematode for example: is composed of only a thousand cells but has about the same number of genes as a human. | | The more complex the organism, the more genetic information is needed (larger c value). | | | | ## Karyotyping * **Definition:** Systematic arrangement of metaphase chromosomes in descending manner. Determined by observing of chromosome number, size and morphology. * **Function:** To detect number of chromosome, size and chromosomal abnormalities. ## Banding techniques * **Definition:** It is a technique of staining which produce staining patterns that are characteristic for each chromosome pair. * **Function:** Provide detection of any structural abnormalities with great accuracy. * **Band:** part of chromosome that is clearly distinguished from adjacent parts by virtue of lighter or darker staining intensity. ## Types of banding The banding techniques can be classified into two main groups: 1. **Bands stained the whole length of chromosome:** it include Q, G, and R bands. 2. **Bands stained the specific part of chromosome:** it include C, T, and NOR bands. 1. **Quinacrine banding (Q- banding):** * It is the first used banding technique. It use quinacrine dye which give fluorescence so it must be examined with **dark field microscope**. The more bright band usually found in regions of chromosome rich in A-T bases while less bright bands is regions rich in C-G bases. * **Advantages:** 1. It is the simplest and the first chromosomal banding method. 2. It gives dark-light banding pattern that is highly characteristic and specific for each chromosome, used to identify particular chromosomes in a cell and structural abnormalities. * **Disadvantages:** 1. It is of temporary pattern. 2. Needs fluorescent microscope. 3. Quinacrine is carcinogenic substance 2. **Giemsa banding (G- banding):** * It give the same banding pattern of Q- banding as dark bands are the A-T rich regions while light bands are C-G rich regions. * **Advantages:** 1. It avoid disadvantages of Q- banding. 2. It is permanent. * **Disadvantages:** 1. In cattle the centromere region is C-G rich regions so appeared light bands so any defect in centromere difficult to be examined. 2. In most of mammalians telomeres is C-G rich regions so appeared light bands so the ends of chromosomes aren't clear and its real length. 3. **Reverse banding (R-banding):** * It reverse of G-band 1. G-C rich regions is dark bands 2. A-T rich regions is light bands. * **Advantages:** 1. It solve the problem of disadvantages of G and Q bands. 2. R-banding is a useful complement to G-banding because some small light G band can be more detected when they are stained by R-banding. 3. R-banding is also useful for visualization of telomere sequence at the ends of chromosomes. Telomeres stained dark with R-banding while light with G-banding. 4. **Centromeric banding (C-banding):** * It stained the regions of centromeres and telomeres of some chromosomes. * **Advantages:** 1. Detect the defects of centromeres. * **Disadvantages:** 1. It detect limited regions of chromosomes (centromeres and telomeres) 5. **Telomeric banding (T-banding):** * It is a modified R-banding technique. * It stained the telomere region of chromosome. 6. **NOR banding** * It stained regions of NOR that contain (rDNA) genes. * Found only in certain specific chromosomes in each species in secondary constriction regions. Not present in all chromosomes and used to identify particular chromosomes. ## Chromosomal aberrations * **Chromosomal aberrations or abnormalities, are changes to the structure or number of chromosomes**, which are strands of condensed genetic material. They usually occur as a result of errors in meiotic / mitotic cell division. They can be inherited from a parent or be de novo. There are two types of chromosomal aberrations: 1. **Numerical chromosomal aberrations:** any Changes in chromosome numbers either decrease or increase of one or more chromosome or even one or more haploid set. 2. **Structural chromosomal aberrations:** changes in normal structure of chromosomes or the arrangement of genetic material within or between individual chromosomes. ## Numerical chromosomal aberrations * **Normally, people are born with 23 chromosome pairs, or 46 chromosomes, in each cell** - one inherited from the mother and one from the father. The normal somatic cell contains two set of chromosomes (2n) (diploid). Normal gamete cell contains one set of chromosomes (1n) (haploid). * **Numerical chromosomal aberrations known as aneuploidy (abnormal number chromosomes).** Usually caused by failure of chromosome division (non-disjunction) which results in cells with an extra chromosome or deficient chromosome. * **The causes of non - disjunction are:** * Aging effect * Radiation * Delayed fertilization after ovulation * **Common numerical abnormalities:** 1. **Triploidy:** * **Triploidy is a rare chromosomal abnormality.** And it is the presence of an additional set of chromosomes in the cell for a total of 69 chromosomes rather than the normal 46 chromosomes per cell. The extra set of chromosomes originates either from the father or the mother during fertilization. It occurs in 1 to 2% of all pregnancies. Most Triploid die early in pregnancy - spontaneous miscarriages (~10%). Almost all other babies die later or are stillborn. Live born very rare. It is not hereditary. There are no specific risk factors. Not more common in older mothers. No increased risk in future pregnancies. failure of meiotic division -2 N gamete + haploid gamete of other parent= Triploid Zygote (69 XXX,69 XXY,69 XYY). 2.**Trisomy:** * **A trisomy is a chromosomal condition characterized by an additional chromosome** (affected person has three copies of one of the chromosomes instead of two). This means they have 47 chromosomes instead of 46. * Down syndrome, Edward syndrome and Patau syndrome are the most common forms of trisomy. All trisomies could be due to the following causes: * nondisjunction * translocation * mosaicism. * **Examples of trisomy:** 1. **Klinefelter syndrome (KS):** is a syndrome where a male has an additional copy of the X chromosome. The primary features are infertility and small, poorly functioning testicles. It is common in cats. sterile male cat which has unusual color (tortoise-shell) that color is normal in heterozygous female as it controlled by codominant X- linked gene as O gives orange color while o gives black color. Karyotype (39, XXY) It has small undeveloped tests, sterile due to no production of sperms, show some feminized characters. 2. **Triple X condition:** It observed in unfertile mare may be sterile but sometimes normal and able to produce normal offspring, when cytologically. Karyotype: (65, XXX). Trisomy in X chromosome. 3. **XYY male condition:** Observed in male human which is taller than 180 cm, mentally retarded, affected with acne and usually tend to be criminal. Karyotype: (47, XYY). 4. **Down syndrome (mongolism):** Karyotype: (47, 21+) Trisomy in autosomal chromosome number 21. The most common autosomal trisomy as one form 700 live birth affected with this case. The main symptoms is Mongol-like eye shape, the affected person is short and have small head, idiotic features, broad hands and mentally retarded. 5. **Patau syndrome:** Karyotype: (47,13+) Trisomy in autosomal chromosome number 13. It is rear condition as one form 20,000 live birth affected with this case. The affected child has several malformations such ads harelip, cleft palate and polydactyl also he is mentally retarded. He Lives less than six months. 6. **Edwards syndrome:** Karyotype (47, XX,18+) Trisomy in chromosome number 18. Occurs in around one in 6,000 live births, and around 80% of those affected are female. The incidence increases as the mother's age increases. The majority of fetuses with the syndrome die before birth. The syndrome has a very low rate of survival, resulting from heart abnormalities, kidney malformations, and other internal organ disorders. 7. **Trisomy in cattle:** Trisomy in chromosome 23 cause Dwarf calves and trisomy in chromosome 17 cause Bachygnathia (very short lower jaw). ## Diagnosis of trisomy conditions * **Prenatal tests that can help detect trisomy disorders include:** * ultrasound scans - sound waves are used to create a picture. * maternal serum screening - a specialized blood test. * amniocentesis - a sample of the amniotic fluid is taken and examined. * chorionic villus sampling - a sample of cells from the chorion, the tissue that will ultimately become the placenta, is taken and examined. * non-invasive prenatal testing (NIPT) - a screening test that measures fetal DNA circulating in the mother's blood. ## Monosomy * **The term "monosomy" is used to describe the absence of one member of a pair of chromosomes.** Therefore, there are 45 chromosomes in each cell of the body instead of the usual 46. In most cases, embryos with monosomy of the autosomes or sex chromosomes are not viable. Some individuals with monosomy of the sex chromosomes (45XO genotype) can survive. * **Example of monosomy:** 1. **Turner syndrome (63, XO)** * is a condition that affects females and involves the partial or complete absence of an X chromosome. This case was discovered in infertile mare. Appearance: generally normal but Estrous cycle is irregular or even absent. Uterus and ovaries is smaller than normal. Ovaries lack to follicles. This case is also reported in mice, rats, pig, horses and also in human. 2. **Mosaicism:** * **Mosaicism is a condition in which cells within the same person have a different genetic makeup.** Results when some of the cells in the body are normal and other cells have a trisomic or monosomic complement eg: trisomy 21 (46 XX / 47 XX + 21) (45% /55%). Mosaicism is caused by an error in cell division very early in the development of the unborn baby. This condition can affect any type of cell, including: * Blood cells * Egg and sperm cells * Skin cells ## N.B: * **Most aneuplodies are incompatible with life resulting in spontaneous abortions except for trisomy 21, 13 and 18 and monosomy X which can result in viable pregnancies.** * **Polyploidy is a heritable condition in which the cells of an organism have more than two paired (homologous) sets of chromosomes.** Most species whose cells have nuclei (eukaryotes) are diploid, meaning they have two sets of chromosomes-one set inherited from each parent. However, some organisms are polyploid, and polyploidy is especially common in plants. Most eukaryotes have diploid somatic cells, but produce haploid gametes (eggs and sperm) by meiosis. * **Polyploidy may occur due to:** * Polyandry fertilization of one ovum by more than one sperm. * Polygyny failure of exclude the one or more polar body and fertilized by one sperm. * **A monoploid has only one set of chromosomes, and the term is usually only applied to cells or organisms that are normally haploid.** Males of bees and other Hymenoptera, for example, are monoploid. ## Structural Chromosomal aberrations * **Structural chromosomal abnormalities are changes in specific part of the chromosome which result from chromosome breakage and reunion in an abnormal way.** Chromosome breakage is caused by X-rays, various chemicals, and can also occur spontaneously. ## Types of structural chromosomal abnormalities: | | | |--------------------------------------------|--------------------------------------------------------------| | **Balanced structural aberrations** | **Unbalanced structural aberrations** | | No change in genetic material and usually give normal phenotype | Increased or decreased genetic material and give abnormal phenotype | | Translocations- inversions | Deletions-insertions-duplications-rings-isochromosomes | ## Unbalanced structural aberrations 1. **Deletions:** * **Definition:** Part of a chromosome is missed or deleted which can make that chromosome less functional and occur during DNA replication (Any number of nucleotides can be deleted, from a single base to an entire piece of chromosome). * **Deletions do not revert, because the DNA is gone (degraded)** * **For example, when part of a short arm in chromosome 5 is deleted, this causes Cri-du-chat syndrome ("cry of the cat")**. Common symptoms of which are reduced head size, high-pitched crying in infants and severe mental retardation and physical abnormalities. * **Causes:** 1. Unequal crossing over 2. Breaking caused due to heat or radiation, Viruses, Chemicals without rejoining 3. Losses from translocation **Types of deletion:** * **Terminal deletion:** a deletion that occurs towards the end of a chromosome. * **Interstitial deletion:** a deletion that occurs from the interior of a chromosome. * **Microdeletion:** are too small to be detected by karyotype. * **Effect of a deletion:** depend on the location and size of the deleted part. * No known living human has an entire autosome deleted from the genome. * A deletion in one allele of a homozygous wild-type organism may give a normal phenotype * While the same deletion in the wild-type allele of a heterozygote would produce a mutant phenotype. * Deletion of the centromere results in an acentric chromosome that is lost. 2. **Duplications:** * **Definition:** Duplications result from doubling of chromosomal segments or the entire chromosome. * **This occurs in Charcot-Marie-Tooth disease type I, which duplicates part of chromosome 17, causing muscle weakness.** * **Causes:** unequal crossing-over (recombination) * **Types** * **Inter-Chromosomal duplication:** The duplicated segment of a chromosome is present in another chromosome of the genome. It is of two types: * **The duplicated segment of a chromosome is incorporated into a non-homologous chromosome.** * **The duplicated segment is present as a separate chromosome.** Clearly, it must have a centromere to be able to survive. * **Intra-Chromosomal duplication:** * **The duplicated segment remains in the same chromosome.** It may be present at different locations * In the other arm. * In the same arm but removed from the original segment. * In the same arm and next to the original segment. This type of duplication is called **tandem duplication** which is further subdivided into the following two types * **Direct tandem:** Gene order of the duplicated segment is the same as that of the original segment. * **Reverse tandem:** Gene order of the duplicated segment is inverted. * **Effect of duplications:** **may produce specific effects when the phenotype is affected due to a change in the position of a gene;** it is called **position effect.** 3. **Ring chromosome:** * **Definition:** Chromosome undergoes 2 breaks and broken ends reunite in a ring. * **Causes:** mutagens like radiation, but they may also arise spontaneously during development. * **Example of human disorders associated with ring chromosome** * **Ring chromosome 14 syndrome is a very rare human.** * **Symptoms:** * Facial abnormalities * Immune deficiencies * Abnormalities of retina * Slow growth * Short stature 4.**Isochromosome:** * **The chromosome consists of two copies of the same arm (Mirror image around centromere)** * **It is due to transverse centromere division instead of normal longitudinal division.** * **The isochromosomes seen mostly involving X - chromosome.** * **Origin of isochromosomes** can be created during mitosis and meiosis through a mis division of the centromere or U-type strand exchange. 5. **Insertions**