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Genetics and Protein Synthesis Felix Hernandez, M.D. The Monk and his peas Gregor Mendel, developed the fundamental principles that would become the modern science of genetics. Mendel demonstrated that heritable properties are parceled out in discrete units, independently inherited. These eventually were termed genes. Mendel's experimental organism was a common garden pea (Pisum sativum), which has a flower that lends itself to selfpollination The Pea Plant Studies The garden peas were planted and studied for eight years. Each character studied had two distinct forms, such as tall or short plant height, or smooth or wrinkled seeds. Mendel's experiments used some 28,000 pea plants. Characteristics Studied Characteristics Studied Mendel’s Contribution Mendel was able to demonstrate that traits were passed from each parent to their offspring through the inheritance of genes. Mendel's work showed: Each parent contributes one factor of each trait shown in offspring. The two members of each pair of factors segregate from each other during gamete formation. The blending theory of inheritance was discounted. Males and females contribute equally to the traits in their offspring. Acquired traits are not inherited. Principle of Segregation A cross involving only one trait is referred to as a monohybrid cross. Mendel crossed pure-breeding (also referred to as true-breeding) smooth-seeded plants with a variety that had always produced wrinkled seeds To help with record keeping, generations were labeled and numbered. The parental generation is denoted as the P1 generation. The offspring of the P1 generation are the F1 generation (first filial). The self-fertilizing F1 generation produced the F2 generation (second filial). Principle of Segregation Mendel's Results Summary of Mendel's Results: The F1 offspring showed only one of the two parental traits, and always the same trait. Results were always the same regardless of which parent donated the pollen. The trait not shown in the F1 reappeared in the F2 in about 25% of the offspring. Traits remained unchanged when passed to offspring: they did not blend in any offspring but behaved as separate units. Reciprocal crosses showed each parent made an equal contribution to the offspring. Mendel's Conclusions Evidence indicated factors could be hidden or unexpressed, these are the recessive traits. The term phenotype refers to the outward appearance of a trait, while the term genotype is used for the genetic makeup of an organism. Male and female contributed equally to the offspring's' genetic makeup: therefore the number of traits was probably two (the simplest solution). Upper case letters are traditionally used to denote dominant traits, lower case letters for recessives. Principle of Independent Assortment Principle of Independent Assortment -that when gametes are formed, alleles assort independently. We now interpret the Principle of Independent Assortment as alleles of genes on different chromosomes are inherited independently during the formation of gametes Punnett squares Step 1 - definition of alleles and determination of dominance. Step 2 - determination of alleles present in all different types of gametes. Step 3 - construction of the square. Step 4 - recombination of alleles into each small square. Step 5 - Determination of Genotype and Phenotype ratios in the next generation. Step 6 - Labeling of generations, for example P1, F1, etc. Punnett squares Genetic Terms Gene - a unit of inheritance that usually is directly responsible for one trait or character. Allele - an alternate form of a gene. Usually there are two alleles for every gene, sometimes as many a three or four. Homozygous - when the two alleles are the same. Heterozygous - when the two alleles are different, in such cases the dominant allele is expressed. Dominant - a term applied to the trait (allele) that is expressed irregardless of the second allele. Recessive - a term applied to a trait that is only expressed when the second allele is the same (e.g. short plants are homozygous for the recessive allele). Phenotype - the physical expression of the allelic composition for the trait under study. Genotype - the allelic composition of an organism. Punnett squares - probability diagram illustrating the possible offspring of a mating. Incomplete dominance Incomplete dominance is a condition when neither allele is dominant over the other. The condition is recognized by the heterozygotes expressing an intermediate phenotype relative to the parental phenotypes. If a red flowered plant is crossed with a white flowered one, the progeny will all be pink. Polygenic Inheritance Polygenic inheritance is a pattern responsible for many features that seem simple on the surface. Many traits such as height, shape, weight, color, and metabolic rate are governed by the cumulative effects of many genes. Polygenic traits are not expressed as absolute or discrete characters polygenic traits are recognizable by their expression as a gradation of small differences (a continuous variation). The results form a bell shaped curve, with a mean value and extremes in either direction. Polygenic Inheritance Usually polygenic traits are distinguished by Traits are usually quantified by measurement rather than counting. Two or more gene pairs contribute to the phenotype. Phenotypic expression of polygenic traits varies over a wide range. Pleiotropy the effect of a single gene on more than one characteristic Sickle-celled individuals suffer from a number of problems, all of which are pleiotropic effects of the sickle-cell allele. Chromosome Abnormalities Chromosome Abnormalities The physical carrier of inheritance four nitrogenous bases: cytosine, thymine, adenine, and guanine; deoxyribose sugar; and a phosphate group. The basic unit (nucleotide) was composed of a base attached to a sugar and that the phosphate also attached to the sugar The Structure of DNA DNA is a double helix, with bases to the center (like rungs on a ladder) and sugarphosphate units along the sides of the helix (like the sides of a twisted ladder). The strands are complementary (deduced by Watson and Crick from Chargaff's data, A pairs with T and C pairs with G, the pairs held together by hydrogen bonds). The Structure of DNA DNA Replication Nucleotides have to be assembled and available in the nucleus, along with energy to make bonds between nucleotides. DNA polymerases unzip the helix by breaking the H-bonds between bases. Once the polymerases have opened the molecule, an area known as the replication bubble forms (always initiated at a certain set of nucleotides, the origin of replication). New nucleotides are placed in the fork and link to the corresponding parental nucleotide already there (A with T, C with G). DNA Replication Since the DNA strands are antiparallel, and replication proceeds in the 5' to 3' direction on EACH strand, one strand will form a continuous copy, while the other will form a series of short Okazaki fragments. DNA Replication One-gene-one-protein One gene codes for the production of one protein. "One gene one enzyme" has since been modified to "one gene one polypeptide" since many proteins (such as hemoglobin) are made of more than one polypeptide. RNA Ribonucleic acid (RNA) was discovered after DNA Information flow (with the exception of reverse transcription) is from DNA to RNA via the process of transcription, and thence to protein via translation. Transcription is the making of an RNA molecule off a DNA template. Translation is the construction of an amino acid sequence (polypeptide) from an RNA molecule Uses of RNA Types of RNA Messenger RNA (mRNA) is the blueprint for construction of a protein. Transfer RNA (tRNA) is the truck delivering the proper amino acid to the site at the right time. RNA has ribose sugar instead of deoxyribose sugar. The base uracil (U) replaces thymine (T) in RNA. Most RNA is single stranded, although tRNA will form a "cloverleaf" structure due to complementary base pairing. Transcription making an RNA copy of a DNA sequence RNA polymerase opens the part of the DNA to be transcribed. Only one strand of DNA (the template strand) is transcribed. RNA nucleotides are available in the region of the chromatin and are linked together similar to the DNA process. Transcription Translation RNA code into protein The code consists of at least three bases To code for the 20 essential amino acids a genetic code must consist of at least a 3-base set (triplet) of the 4 bases 64 possibilities The genetic code consists of 61 amino-acid coding codons and three termination codons, which stop the process of translation. The genetic code is thus redundant (degenerate in the sense of having multiple states amounting to the same thing), with, for example, glycine coded for by GGU, GGC, GGA, and GGG codons. Translation Protein Synthesis Promoters are sequences of DNA that are the start signals for the transcription of mRNA. Terminators are the stop signals Transfer RNA (tRNA) is basically cloverleaf-shaped. tRNA carries the proper amino acid to the ribosome when the codons call for them. At the top of the large loop are three bases, the anticodon, which is the complement of the codon. There are 61 different tRNAs, each having a different binding site for the amino acid and a different anticodon. For the codon UUU, the complementary anticodon is AAA. Amino acid linkage to the proper tRNA is controlled by the aminoacyl-tRNA synthetases. Energy for binding the amino acid to tRNA comes from ATP conversion to adenosine monophosphate (AMP). Protein Synthesis Translation is the process of converting the mRNA codon sequences into an amino acid sequence. The initiator codon (AUG) codes for the amino acid N-formylmethionine (f-Met). No transcription occurs without the AUG codon. f Met is always the first amino acid in a polypeptide chain, although frequently it is removed after translation. The initiator tRNA/mRNA/small ribosomal unit is called the initiation complex. The larger subunit attaches to the initiation complex. After the initiation phase the message gets longer during the elongation phase. Protein Synthesis Protein Synthesis New tRNAs bring their amino acids to the open binding site on the ribosome/mRNA complex, forming a peptide bond between the amino acids. The complex then shifts along the mRNA to the next triplet, opening the A site. The new tRNA enters at the A site. When the codon in the A site is a termination codon, a releasing factor binds to the site, stopping translation and releasing the ribosomal complex and mRNA. Protein Synthesis Protein Synthesis Often many ribosomes will read the same message, a structure known as a polysome forms. In this way a cell may rapidly make many proteins. The Eukaryotic Genome We use the term genome to refer to all of the alleles possessed by an organism Almost half the DNA in eukaryotic cells is repeated nucleotide sequences. Protein-coding sequences are interrupted by non-coding regions. Non-coding interruptions are known as intervening sequences or introns. Most, but not all structural eukaryote genes contain introns. Although transcribed, these introns are excised (cut out) before translation Coding sequences that are expressed are exons. The Eukaryotic Genome