Noncoding DNA and Phenotypic Differences

Transcriptomics and Proteomics

• Transcriptomics: Characterizing and quantifying mRNAs produced by cells, tissues, organisms, and communities.

  • Tissue samples are taken from individuals of different genotypes.

  • RNA is isolated, including mRNA.

  • Reverse transcriptase makes cDNA copies of mRNA.

  • cDNA is amplified using PCR.

  • cDNA is sequenced, producing reads.

  • Reads are aligned to genome sequence data using software.

  • The number of reads aligned to each gene represents the expression level of that gene.

  • Quantities of each gene's mRNA are displayed relative to a control treatment using a heat map.

    • Green: Highly expressed.

    • Black: Expressed at the same levels as the control.

    • Red: Expressed at low levels.

• Proteomics: Analysis of protein expression.

  • Protein extracts are made from tissue samples.

  • Proteins are separated by size using gel electrophoresis.

  • Specific proteins can be detected using antibodies in Western blot or immunoblot methods.

  • Differences in band intensity represent differences in protein amounts.

  • Digital imaging quantifies protein concentrations.

• Expression patterns of mRNA and protein can be visualized in biological materials.

  • mRNA visualization: A complementary RNA or DNA sequence (probe) binds to the mRNA and is stained blue.

  • Protein visualization: Antibodies specific to the proteins are applied, and chemical staining is used to detect antibody binding.

  • Fluorescent reagents can also be used to visualize mRNA and protein expression.

Case Study 3: Branching in Corn Plants

• Focus: Role of noncoding DNA in influencing the form of corn plants.

• Contrast with previous case studies (Huntington's disease and sheep oral): In this case, coding DNA differences are not important; noncoding DNA differences play causal roles.

• Comparison of Teosinte and Maize:

  • Teosinte: Highly branched with many small ears.

  • Maize: Few branches with only a couple of ears.

• Teosinte branch 1 (tb1) gene:

  • Encodes a transcriptional regulatory protein (TB1).

  • TB1 represses the transcription of genes required for outgrowth to develop from the main stem, and therefore, branching.

  • The TB1 protein does not differ substantially in structure or function between maize and teosinte. It represses transcription of genes required for branching in both species.

• Key Difference: Level of expression of TB1.

  • Teosinte: TB1 is expressed at low levels in the developing stem.

    • Transcription of genes required for branching is not repressed.

    • Branching genes are expressed at high levels, resulting in many side branches.

  • Maize: TB1 is expressed at high levels in the developing stem.

    • Transcription of genes required for branching is repressed.

    • Branching genes are expressed at very low levels or not at all, resulting in few or no side branches.

• Variants of the TB1 gene that are expressed at high levels arose by mutation in a Teosinte population in Mexico approximately 9,000 years ago.

Maize vs. Teosinte: Differences and Similarities

• The key difference between Maize and Teosinte TB1 alleles is in the noncoding DNA, which contains the regulatory switches.

Examples of Branching

• Sunflowers that had their main stalks eaten off by deer developed multiple flowers on side branches instead of one flower on the main stalk.

Case Study: Lactose Tolerance in Humans

• Background:

  • Infants and children can typically digest milk (lactose).

  • As adults, some people can digest milk (lactose tolerance), while others cannot (lactose intolerance).

• Lactose: Main sugar in milk, broken down by the enzyme lactase in the intestines.

• Lactase: Encoded by the lactase gene on chromosome two.

• Phenomena to Explain:

  • Variation in the ability to digest dairy products during an individual's lifetime.

  • Variation between different adult individuals in the ability to digest dairy products.

Key Concepts

Genes and Variation:

• Lactase gene

• Allele

• DNA sequence differences

• Genome

• Coding DNA

• Noncoding DNA

• Promoter sequence

• Gene regulatory switches

Transcription:

• Transcription

• Lactase mRNA

• Transcriptional regulatory proteins (TRPs)

• Level of transcription

Proteins and Phenotypes:

• Translation

• Lactase enzyme

• Ability to digest dairy products

• The lactase gene is a sequence of DNA on a chromosome. Each individual has two copies of that chromosome, each containing an allele of the gene.

• Like all protein-coding genes, the lactase gene contains both coding DNA and noncoding DNA.

• Different alleles have different DNA sequences in their coding DNA and/or their noncoding DNA.

• The noncoding DNA contains the promoter sequence and the gene regulatory switches.

• Protein DNA undergoes transcription, resulting in the production of mRNA.

• The mRNA undergoes translation, resulting in the production of the lactase enzyme, which is a protein.

• The enzyme determines the ability to digest dairy products.

Role of Noncoding DNA

• The promoter sequence in the noncoding DNA is where RNA polymerase binds to initiate transcription.

• TRPs bind to the gene regulatory switches in the regulation of transcription.

• This affects the level of transcription and the level of mRNA that is produced, and indirectly the level of protein that is produced.

• For lactase, transcription only occurs in brush border cells in the lining of the small intestine because of the function of transcriptional regulatory proteins found only in those cells.

• The gene regulatory switches of the lactase gene cause the gene to be switched on in infants and children.

• In many adult individuals, the transcription of the gene gets turned off because a different set of gene regulatory switches is used for activation of transcription of the lactase gene in adults and children. In many adults, there are no functional gene regulatory switches for adult expression of lactase.

Adult Gene Regulatory Switches

• In the human population, there is variation in the DNA sequences of these adult gene regulatory switches.

• These differences cause some individuals to continue to be able to transcribe the lactase gene during adulthood.

• In other individuals, the adult gene regulatory switches are not functional, and transcription of lactase is not able to be activated.

Lactose Tolerance Causes

Childhood:

• Both person one and person two could digest milk because at one or both alleles of their lactase genes, TRPs in their intestinal lining cells were able to bind to gene regulatory switches to activate transcription of the gene.

• Lactase gene was transcribed, and mRNA was translated to make the lactase enzyme.

Adulthood:

• A different set of gene regulatory switches is utilized to activate transcription of the lactase gene.

• Person one, the adult gene regulatory switches are functional, and they can digest milk.

• Person two the adult gene regulatory switches are not functional, and transcription of the lactase gene cannot be activated.

Comparison Between Cases

• Different cell types in the same individual.

• Different developmental stages of the same individual.

• All of these cases, the body cells have the same DNA sequence.

• The gene regulatory switches are the same between different cell types and in all the body cells of a persons drug development and a butterfly's drug development.

• Different cell types of the same person utilize their gene regulatory switches differently.

• The different transcription of genes between cell types leads to differences in the pools of mRNA that are produced by the different cell.

• The protein expression differences can be characterized as in the proteomic methods we talked about at the beginning of the lecture.

• If we have differences in gene regulatory switches of a gene between individuals, as we talked about in the corn branching case with t b one, we can see how noncoding DNA plays a major causal role in the differences in phenotype among individuals of the same species and between other species.

Conclusion

This lecture illustrates how this one powerful big idea, the noncoding DNA contains information that is used by cells to express genes differently, producing differences in structure and function, forms a critical part of the explanation for a huge diversity of biological forms and phenotypes that we see among different individuals of the same species and among different species in nature.

• Transcriptomics: Detailed characterization and quantification of all mRNA molecules produced within a cell, tissue, organism, or community. This involves:

  • Taking tissue samples from individuals with different genotypes or under different conditions.

  • Isolating RNA, specifically focusing on mRNA, which carries genetic information from DNA to ribosomes.

  • Using reverse transcriptase to create cDNA (complementary DNA) copies of the mRNA, making it more stable for further analysis.

  • Amplifying the cDNA using PCR (polymerase chain reaction) to increase the quantity of DNA for sequencing.

  • Sequencing the amplified cDNA to generate reads, which are short sequences of DNA.

  • Aligning these reads to a reference genome sequence using bioinformatics software to determine the origin and expression level of each gene.

  • Quantifying the number of reads that align to each gene, which represents the expression level of that gene. Higher read counts indicate higher expression levels.

  • Displaying the quantities of each gene's mRNA relative to a control treatment using a heat map. Heat maps provide a visual representation of gene expression levels, typically:

    • Green: Indicates genes that are highly expressed compared to the control.

    • Black: Indicates genes expressed at the same levels as the control.

    • Red: Indicates genes expressed at low levels compared to the control.

• Proteomics: Comprehensive analysis of protein expression within a sample, including:

  • Preparing protein extracts from tissue samples to isolate proteins for study.

  • Separating proteins by size using gel electrophoresis, which involves applying an electric field to move proteins through a gel matrix.

  • Detecting specific proteins using antibodies in Western blot or immunoblot methods, where antibodies bind to target proteins and allow for their detection.

  • Quantifying differences in band intensity on the gel, which represent differences in protein amounts. Denser bands indicate higher protein concentrations.

  • Using digital imaging techniques to quantify protein concentrations, providing precise measurements of protein abundance.

• Visualizing mRNA and Protein Expression Patterns:

  • mRNA visualization: Use of complementary RNA or DNA sequences (probes) that bind to the mRNA of interest, followed by staining to make the location visible (often blue staining).

  • Protein visualization: Application of antibodies specific to the proteins of interest, followed by chemical staining to detect antibody binding, indicating the location and abundance of the protein.

  • Use of fluorescent reagents to visualize both mRNA and protein expression, allowing for high-resolution imaging and quantification.

Case Study 3: Branching in Corn Plants

• Focus: Understanding the role of noncoding DNA in determining the form of corn plants.

• Contrast: Unlike previous cases where coding DNA differences were significant, here, noncoding DNA differences play a crucial role.

• Comparison of Teosinte and Maize:

  • Teosinte: Characterized by extensive branching and numerous small ears.

  • Maize: Characterized by few branches and only a couple of ears.

• Teosinte branch 1 (tb1) gene:

  • Encodes a transcriptional regulatory protein (TB1) that controls branching.

  • TB1 represses the transcription of genes required for the outgrowth of branches from the main stem.

  • The TB1 protein itself is highly conserved between maize and teosinte, maintaining similar structure and function in both species.

• Key Difference: Level of expression of TB1:

  • Teosinte: TB1 is expressed at low levels in the developing stem, leading to:

    • Reduced repression of branching genes.

    • High expression of branching genes, resulting in numerous side branches.

  • Maize: TB1 is expressed at high levels in the developing stem, leading to:

    • Strong repression of branching genes.

    • Low or absent expression of branching genes, resulting in few or no side branches.

• The high expression variants of the TB1 gene emerged through mutation in a Teosinte population in Mexico around 9,000 years ago, contributing to the domestication of maize.

Maize vs. Teosinte: Differences and Similarities

• The crucial distinction lies in the noncoding DNA of TB1, which contains regulatory switches that control the level of gene transcription.

Examples of Branching

• Sunflowers that have their main stalks consumed by deer often develop multiple flowers on side branches, demonstrating the plasticity of branching patterns.

Case Study: Lactose Tolerance in Humans

• Background:

  • Infants and children can typically digest lactose due to the production of the enzyme lactase.

  • As adults, some individuals retain the ability to digest lactose (lactose tolerance), while others lose this ability (lactose intolerance).

• Lactose: The primary sugar found in milk, which requires the enzyme lactase to be broken down into simpler sugars for absorption in the intestines.

• Lactase: The enzyme responsible for breaking down lactose, encoded by the lactase gene located on chromosome two.

• Phenomena to Explain:

  • Why the ability to digest dairy products varies among individuals throughout their lifetimes.

  • Why there are differences in the ability to digest dairy products among different adult individuals.

Key Concepts

Genes and Variation:

• Lactase gene: The gene that codes for the production of lactase.

• Allele: Different versions of the lactase gene, which can affect the ability to digest lactose.

• DNA sequence differences: Variations in the DNA sequence of the lactase gene and its regulatory regions.

• Genome: The entire set of genetic material in an organism.

• Coding DNA: The portion of DNA that contains instructions for making proteins.

• Noncoding DNA: The portion of DNA that does not code for proteins but contains regulatory elements.

• Promoter sequence: A region of DNA where RNA polymerase binds to initiate transcription.

• Gene regulatory switches: DNA sequences that control when and where a gene is transcribed.

Transcription:

• Transcription: The process of creating mRNA from a DNA template.

• Lactase mRNA: The messenger RNA molecule that carries the genetic code for lactase.

• Transcriptional regulatory proteins (TRPs): Proteins that bind to gene regulatory switches and control the rate of transcription.

• Level of transcription: The rate at which a gene is transcribed into mRNA.

Proteins and Phenotypes:

• Translation: The process of creating a protein from an mRNA template.

• Lactase enzyme: The protein that breaks down lactose.

• Ability to digest dairy products: The phenotypic trait determined by the activity of the lactase enzyme.

• Each individual has two copies of each chromosome, each containing an allele of the lactase gene.

• The lactase gene contains coding DNA that provides instructions for making the lactase enzyme, and noncoding DNA that regulates when and where the gene is expressed.

• Different alleles of the lactase gene may have different DNA sequences in their coding and noncoding regions, leading to variations in lactase production and activity.

• The noncoding DNA includes the promoter sequence, where RNA polymerase binds to initiate transcription, and gene regulatory switches, which control the level of transcription.

• During transcription, the DNA sequence of the lactase gene is copied into mRNA.

• The mRNA then undergoes translation, resulting in the production of the lactase enzyme, which is a protein that breaks down lactose.

• The activity of the lactase enzyme determines an individual's ability to digest dairy products.

Role of Noncoding DNA

• The promoter sequence in the noncoding DNA is the site where RNA polymerase binds to initiate transcription of the lactase gene.

• Transcriptional regulatory proteins (TRPs) bind to gene regulatory switches in the noncoding DNA to regulate transcription.

• This binding can either enhance or inhibit transcription, affecting the level of mRNA produced and, consequently, the level of lactase enzyme generated.

• Lactase transcription occurs specifically in brush border cells of the small intestine due to the presence of TRPs found only in those cells.

• Gene regulatory switches control the activation of the lactase gene in infants and children, ensuring lactase production during early development.

• In many adult individuals, transcription of the lactase gene is turned off because the gene regulatory switches responsible for adult expression are not functional.

Adult Gene Regulatory Switches

• There is variation in the DNA sequences of adult gene regulatory switches within the human population.

• These sequence differences can cause some individuals to continue transcribing the lactase gene into adulthood, leading to lactose tolerance.

• In other individuals, non-functional adult gene regulatory switches prevent the activation of lactase transcription, resulting in lactose intolerance.

Lactose Tolerance Causes

Childhood:

• Both lactose-tolerant and lactose-intolerant individuals can typically digest milk during childhood because TRPs in their intestinal lining cells can bind to gene regulatory switches, activating transcription of the lactase gene.

• This transcription leads to mRNA production, which is then translated into the lactase enzyme, enabling lactose digestion.

Adulthood:

• A different set of gene regulatory switches becomes responsible for activating transcription of the lactase gene.

• In lactose-tolerant individuals, these adult gene regulatory switches remain functional, allowing continued lactose digestion.

• In lactose-intolerant individuals, the adult gene regulatory switches are non-functional, preventing the activation of lactase gene transcription.

Comparison Between Cases

• These cases illustrate differences in gene expression in different cell types in the same individual and at different developmental stages.

• While all body cells have the same DNA sequence, gene regulatory switches can be utilized differently.

• Differences in transcription of genes between cell types lead to variations in mRNA pools, which result in protein expression differences that can be characterized using proteomic methods.

• Variations in gene regulatory switches among individuals, as demonstrated in the corn branching case with tb1, highlight the significant role of noncoding DNA in phenotypic differences within and between species.

Conclusion

Noncoding DNA contains vital information used by cells to express genes differently, leading to differences in structure and function. This plays a critical role in the diversity of biological forms and phenotypes observed among individuals of the same species and among different species in nature.