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3.E: Isolating and Analyzing Genes (Exercises) - Biology

3.E: Isolating and Analyzing Genes (Exercises) - Biology


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3.2 Altering the ends of DNA fragments for ligation into vectors.

(Adapted from POB)

a) Draw the structure of the end of a linear DNA fragment that was generated by digesting with the restriction endonuclease EcoRI. Include those sequences remaining from the EcoRI recognition sequence.

b) Draw the structure resulting from the reaction of this end sequence with DNA polymerase I and the four deoxynucleoside triphosphates.

c) Draw the sequence produced at the junction if two ends with the structure derived in (b) are ligated.

d) Design two different short synthetic DNA fragments that would permit ligation of structure (a) with a DNA fragment produced by a PstI restriction digest. In one of these synthetic fragments, design the sequence so that the final junction contains the recognition sequences for both EcoRI and PstI. Design the sequence of the other fragment so that neither the EcoRI nor the PstI sequence appears in the junction.

3.3. What properties are required of vectors used in molecular cloning of DNA?

3.4. A student ligated a BamHI fragment containing a gene of interest to a pUC vector digested with BamHI, transformed E. coliwith the mixture of ligation products and plated the cells on plates containing the antibiotic ampicillin and the chromogenic substrate X‑gal. Which colonies should the student pick to find the ones containing the recombinant plasmid (with the gene of interest in pUC)?

3.5. Starting with an isolated mRNA, one wishes to make a double stranded copy of the mRNA and insert it at the PstI site of pBR322 via G-C homopolymer tailing. One then transforms E. coliwith this recombinant plasmid, selecting for tetracycline resistance. What are the four enzymatic steps used in preparing the cDNA insert? Name the enzymes and describe the intermediates.

3.6 A researcher needs to isolate a cDNA clone of giraffe actin mRNA, and she knows the size (Mr = 42,000) and partial amino acid sequence of giraffe actin protein and has specific antibodies against giraffe actin. After constructing a bank of cDNA plasmids from total mRNA of giraffe fibroblasts (dG-dC tailed into the PstI site of pBR322), what methods of screening the bank could be used to identify the actin cDNA clone?

3.7 The restriction map of pBR322 is

The distance in base pairs between restriction sites is as follows:

PstI to EcoRI 750 bp

EcoRI to HindIII 50 bp

HindIII to BamHI 260 bp

BamHI to PstI 3300 bp

A recombinant cDNA plasmid, pAlc-1, has double-stranded cDNA inserted at the PstI site of pBR322, using a technique that retains this cleavage site at both ends of the insert. Digestion of pBR322 and pAlc-1 with restriction endonucleases gives the following pattern after gel electrophoresis (left). The sizes of the fragments are given in base pairs. The DNA fragments were transferred out of the gel onto nitrocellulose and hybridized with radiolabeled cDNA from wild-type A. latrobus(a Southern blot-hybridizaton). Hybridizing fragments are shown in the autoradiogam diagram on the right.

a) What is the size of the cDNA insert?

b) What two restriction endonucleases cleave within the cDNA insert?

c) For those two restriction endonucleases, each DNA fragment in the single digest is cut by PstI into two DNA fragments in the double digest (i.e. the restriction endonuclease plus PstI). Determine which fragments each single digest fragment is cut into, and use this information to construct a map.

d) Draw a restriction map for pAlc-1, showing sites for PstI, EcoRI, BamHI and HindIII. Indicate the distance between sites and show the cDNA insert clearly.

3.8. You isolate and clone a KpnI fragment from A. latrobusgenomic DNA that encodes the mRNA cloned in pAlc-1 (as analyzed in question 3.7). The restriction map of the genomic fragment is

Each fragment that hybridizes to pAlc-1 is indicated by an asterisk. What does this map, especially when compared to that in problem 3.7, tell you about the structure of the gene? Be as quantitative as possible.

3.9. Some particular enzyme is composed of a polypeptide chain of 192 amino acids. The gene that encodes it has 1,440 nucleotide pairs. Explain the relationship between the number of amino acids in this polypeptide and the number of nucleotide pairs in its gene.

3.10. When viewed in the electron microscope, a hybrid between a cloned giraffe actin gene (genomic DNA) and mature actin mRNA looks like this:

What can you conclude about actin gene structure in the giraffe?

3.11. DNA complementary to pepper mRNA was synthesized using oligo (dT) as a primer for first strand synthesis. The second strand (synonymous with the mRNA) was then synthesized, and the population of double stranded cDNAs were ligated into a plasmid vector using a procedure that leaves PstI sites flanking the cDNA insert (i.e. the terminal PstI sites for each clone are not part of the cDNA). This cDNA library was screened for clones made from the mRNA from the pepper yellow gene. One clone was isolated, and subsequent analysis of the pattern of restriction endonuclease cleavage patterns showed it had the following structure:

The map shows the positions of restriction endonuclease cleavage sites and the distance between them in kilobases (kb). The map of the cDNA insert is shown with solid lines, and plasmid vector DNA flanking the cDNA is shown as dotted lines. The top strand is oriented 5' to 3' from left to right, and the bottom strand is oriented 5' to 3' from right to left. The positions and orientations of two oligonucleotides to prime synthesis for sequence determination are shown, and are placed adjacent to the strand that will be synthesized in the sequencing reaction.

a) Oligonucleotides that anneal to the plasmid vector sequences that flank the duplex cDNA insert were used to prime synthesis of DNA for sequencing by the Sanger dideoxynucleotide procedure. A primer that annealed to the vector sequences to the left of the map shown above generated the sequencing gel pattern shown below on the left. A primer that annealed to the vector sequences to the right of the map shown above generated the sequencing gel pattern shown below on the right. The gels were run from the negative electrode at the top to the positive electrode at the bottom, and the segment presented is past the PstI site (i.e. do not look for a PstI recognition site).

Left primer Right primer

G

A

T

C

G

A

T

C

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a) What is the DNA sequence of the left and right ends of the insert in the cDNA clone? Be sure to specify the 5' to 3' orientation, and the strand (top or bottom) whose sequence is reported. The terms left, right, top and bottom all refer to the map shown above for the cDNA clone.

b) Which end of the cDNA clone (left or right in the map above) is most likely to include the sequence synonymous with the 3' end of the mRNA?

c) What restriction endonuclease cleavage sites do you see in the sequencing data given?

3.12. Genomic DNA from the pepper plant was ligated into EcoRI sites in a l phage vector to construct a genomic DNA library. This library was screened by hybridization to the yellowcDNA clone. The pattern of EcoRI cleavage sites for one clone that hybridized to the yellowcDNA clone was analyzed in two experiments.

In the first experiment, the genomic DNA clone was digested to completion with EcoRI, the fragments separated on an agarose gel, transferred to a nylon filter, and hybridized with the radioactive yellowcDNA clone. The digest pattern (observed on the agarose gel) is shown in lane 1, and the pattern of hybridizing fragments (observed on an autoradiogram after hybridization) is shown in lane 2. Sizes of the EcoRI fragments are indicated in kb. The right arm of this l vector is 6 kb long, and the left arm is 30 kb.

In the second experiment, the genomic DNA clone was digested with a range of concentrations of EcoRI, so that the products ranged from a partial digest to a complete digest. The cleavage products were annealed to a radioactive oligonucleotide that hybridized only to the right cohesive end (cossite) of the l vector DNA. This simply places a radioactive tag at the right end of all the products of the reaction that extend to the right end of the l clone (partial or complete); digestion products that do not include the right end of the l clone will not be seen. The results of the digestion are shown above, on the right. Lane 1 is the clone of genomic DNA in l that has not been digested, lane 5 is the complete digest with EcoRI, and lanes 2, 3 and 4 are partial digests using increasing amounts of EcoRI. The sizes of the radioactive DNA fragments (in kb) are given, and the density of the fill in the boxes is proportional to the intensity of the signal on the autoradiogram.

a) What is the map of the EcoRI fragments in the genomic DNA clone, and which fragments encode mRNA for the yellowgene? You may wish to fill in the figure below; the left and right arms of the l vector are given. Show positions of the EcoRI cleavage sites, distances between them (in kb) and indicate the fragments that hybridize to the cDNA clone.

EcoRI EcoRI

Left arm ___| |_ Right arm

(30 kb) (6 kb)

In a third experiment, the pepper DNA from the genomic DNA clone was excised, hybridized with yellowmRNA under conditions that favor RNA-DNA duplexes and examined in the electron microscope to visualize R-loops. A pattern like the following was observed. The lines in the figure can be duplex DNA, RNA-DNA duplexes and single-stranded DNA.

b) What do the R-loop data indicate? Please draw an interpretation of the R-loops, showing clearly the two DNA strands and the mRNA and distinguishing between the template (bottom, or message complementary) and nontemplate (top, or message synonymous) strands.

The EcoRI fragments that hybridize to the yellowcDNA clone were isolated and digested with SalI (S in the figure below), HindIII (H), and the combination of SalI plus HindIII (S+H). The resulting patterns of DNA fragments are shown below; all will hybridize to the yellowcDNA clone. Cleavage of the 5 kb EcoRI fragment with SalI generates two fragments of 2.5 kb.

c) What are the maps of the SalI and HindIII site(s) in each of the EcoRI fragments? Show positions of the cleavage sites and distances between them on the diagram below.

5 kb EcoRI fragment: 4 kb EcoRI fragment:

EcoRI EcoRI EcoRI EcoRI

|___________________________| |___________________________|

d) Compare these restriction maps with that of the cDNA clone (problem 1.38) and the R-loops shown above. Assuming that the SalI and HindIII sites in the genomic DNA correspond to those in the cDNA clone, what can you deduce about the intron/exon structure of the yellowgene(s) contained within the 5 kb and 4 kb EcoRI fragments? Please diagram the exon-intron structure in as much detail as the data permit (i.e. show the size of the intron(s) and positions of intron/exon junctions as precisely as possible).

5 kb EcoRI fragment: 4 kb EcoRI fragment:

EcoRI EcoRI EcoRI EcoRI

|___________________________| |___________________________|

e) Considering all the data (maps of cDNA and genomic clones and R-loop analysis), what can you conclude about the number and location(s) of yellowgene(s) in this genomic clone?

3.13 You have isolated an 1100 base pair (bp) cDNA clone for a gene called azure that when mutated causes blue eyes in frogs. You also isolate a 3000 bp SalI genomic DNA fragment that hybridizes to the azurecDNA. The map of the azure cDNA is as follows, with sizes of fragments given in bp.

Digestion of the 3000 bp SalI fragment of genomic DNA with the indicated restriction endonucleases yields the following pattern of fragments, all of which hybridize to the azurecDNA. Remember that the starting fragment has SalI sites at each end. Sizes of fragments are in bp.

Restriction enzymes

BamHI Bam+Pst PstI Pst+Eco EcoRI Bam+Eco

2700

2300

2000

1900 1900

1200

1100 1100

800

700 700 700

300 300 300

The SalI to SalI (3000 bp) genomic fragment was hybridized to the 1100 bp cDNA fragment, and the heteroduplexes were examined in the electron microscope. Measurements on a large number of molecules resulted in the determination of the sizes indicated in the structure on the left, i.e. duplex regions of 400 and 600 bp are interrupted by a single stranded loop of 1500 nucleotides and are flanked by single stranded regions of 500 and 100 nucleotides. When the same experiment is carried out with the 2700 bp SalI to EcoRI genomic DNA fragment hybridized to the cDNA fragment, the structure on the right is observed.


a) What is the restriction map of the 3000 bp SalI to SalI genomic DNA fragment from the azuregene? Specify distances between sites in base pairs.

b) How many introns are present in the azuregenomic DNA fragment?

c) Where are the exons in the azuregenomic DNA fragment? Draw the exons as boxes on the restriction map of the 3000 bp SalI to SalI genomic DNA fragment? Specify (in base pairs) the distances between restriction sites and the intron/exon boundaries.

3.14 The T-cell receptor is present only on T-lymphocytes, not on B-lymphocytes or other cells. Describe a strategy to isolate the T-cell receptor by subtractive hybridization, using RNA from T-lymphocytes and from B-lymphocytes.

3.15.How many exons are in the human insulin (INS) gene, how big are they, and how large are the introns that separate them? Use three different bioinformatic approaches to answer this.

a. Align the available genomic sequence containing INS(encoding insulin) with the sequence of the mRNA to find exons and introns in the INSgene. The sequence files are:

INSmRNA: accession number NM_000207

INS gene (includes part of THand IGF2in addition to INS): accession number L15440

Files can be obtained from NCBI (http://www.ncbi.nlm.nih.gov), or from the course web site (www.bmb.psu.edu/Courses/bmb400/default.htm)

Align the mRNA (cDNA) and genomic sequence using the BLAST2sequences server at

and the sim4server at

pbil.univ-lyon1.fr/sim4.html

Sim4is designed to take into account terminal redundancy at the exon/intron junctions, whereas BLAST2does not. Do you see this effect in the output?

b. Use the ab initioexon finding program Genscan, available at

genes.mit.edu/GENSCAN.html

to predict exons in the INSgenomic sequence (L15440).

How does this compare with the results of analyzing with the program genscan?

c. What do you see for INSat the Human Genome Browser and Ensembl? They are accessed at:


Isolation of Caenorhabditis elegans genomic DNA and detection of deletions in the unc-93 gene using PCR

PCR, genomic DNA isolation, and agarose gel electrophoresis are common molecular biology techniques with a wide range of applications. Therefore, we have developed a series of exercises employing these techniques for an intermediate level undergraduate molecular biology laboratory course. In these exercises, students isolate genomic DNA from the nematode Caenorhabditis elegans and use PCR to detect deletions in the C. elegans unc-93 gene. In advance of the exercises, wild-type and three different unc-93 deletion mutant strains are grown, harvested, and frozen by the instructor. In one approach, students isolate genomic DNA from each strain using a genomic DNA isolation kit and use agarose gel electrophoresis to analyze the DNA and to estimate its concentration. PCRs using primers directed to two different regions of the unc-93 gene are carried out on the genomic DNA from wild-type and mutant strains, and the PCR products are analyzed by agarose gel electrophoresis. Students analyze the gel to determine the approximate location and size of deletions in the three mutant strains. Alternatively, students may lyse single nematodes and carry out PCR in one laboratory session. These exercises should be easily adaptable to detection of well characterized deletions in any organism.


Gene trap expression and mutational analysis for genes involved in the development of the mammalian nervous system †

A. Stoykova, K. Chowdhury, P. Bonaldo, and M. Torres all contributed equally to this paper.

Abstract

We have used a large-scale gene-trap approach for the isolation and mutation of genes that might play roles in the developing nervous system. After in vitro integration of two different gene trap vectors (pGT1.8geo: Skarnes et al. [1995] Proc. Natl. Acad. Sci. USA 92:6592–6596 IRESβgeo: Chowdhury et al. [1997] Nucleic Acids Res. 25:1531–1536) in mouse embryonic stem (ES) cell lines, we created 64 transgenic mouse lines. The expression analysis of the reporter gene during embryogenesis of heterozygous embryos revealed 47 lines with a variety of patterns. Around one-third (36%) of these gene trap lines showed spatiotemporal expression that was either restricted predominantly in the developing nervous system (11 lines 17%) or widespread but with very high levels of expression in the nervous tissue (12 lines 19%). In most cases, a correlation was found between the in vitro and the in vivo patterns of the reporter gene expression. Thus far, preliminary mutant analysis of 16 gene trap lines with potentially interesting expression patterns in the developing nervous system showed that mice homozygous for eight (50%) insertions were lethal, whereas the homozygous mice from five gene trap lines (31%) showed a lower than expected Mendelian ratio of live homozygous animals. Analysis of β-galactosidase reporter gene expression during embryogenesis has shown that four transgenic lines are useful lacZ in situ markers for specific regions of the developing nervous system. Here, we discuss some in vivo and in vitro selection criteria that may increase the number of the trapped genes potentially involved in the control of neural development and some future strategies to improve further the efficiency of the gene trap approach. Dev. Dyn. 1998212:198–213. © 1998 Wiley-Liss, Inc.


Online Resources

The bioinformatics applications used in the exercises detailed below are open-access and web-based, with user-friendly interfaces that run in common web browsers of PC and Mac computers. Although the applications chosen are hosted in long-established web-based platforms that are widely used and currently indispensable in daily research routines, it is important to instruct students about the evolving dynamics of these bioinformatics applications, resulting from the addition of more data, the development of new resources, or the display of increasingly intuitive interfaces. A pilot trial of these bioinformatics activities was carried out in a classroom setting with the collaboration of 14 teachers from six schools and involving a total of 387 high school students (15–18 years old).


3.E: Isolating and Analyzing Genes (Exercises) - Biology

1. A gene pool consists of all the genes and their different alleles, present in an interbreeding population.

  • gene pool = all the genes in an interbreeding population at a certain time.
  • allele frequency = the frequency of an allele, as a proportion of all the alleles of of a gene, in a population.
    • allele frequencies range from 0 to 1.0, or as a percentage
    • evolution always involves a change in allele frequency in a population's gene pool, over a number of generations

    Skill: Comparison of allele frequencies of geographically isolated populations.

    2. Evolution requires that allele frequencies change with time in populations.

    3. The biological species concept is based on reproductive isolation of a gene pool.

    • prior to Darwin, each species was regarded as a fixed entity, morphologically distinct from other species
      • Darwin recognized that the reason individuals within a species are morphologically similar is because they interbreed
      • the ability to interbreed is more important than morphological characteristics
      • biological species definition: a group of a potentially interbreeding populations, with a common gene pool, which are reproductively isolated from other such groups
      • sibling species are populations that cannot interbreed, but are morphologically indistinct
      • some pairs of species are morphologically different, but do interbreed
      • many plant species form hybrids
      • some species always reproduce asexually
      • fossil species cannot be classified according to the biological species definition

      4. Application: Identifying examples of directional, stabilizing and disruptive selection.

      5. Reproductive isolation of populations can be temporal, behavioural or geographic.

      • When two populations of a species are segregated by a geographic barrier such that they cannot reproduce, they are considered allopatric.
      • Dispersal mechanisms cause some individuals of a population to migrate to new locations, separating the parental, sympatric population, into two or more allopatric populations
      • allopatric populations that are reproductively isolated will diverge due to:
        • differing natural selection pressures due to slightly differing environments
        • differing mutation pressures due to the random nature of mutation
        • genetic drift, as chance is likely to produce slightly different allelic frequencies in the allopatric populations, especially if the number of founding members of the population is few.
        • Sympatric populations overlap in geographic distribution
        • a portion of a population may become fixed on resources not used by the parent population
        • differences in resource use can lead to a balance between two different adaptations, a balanced polymorphism
        • if the balanced polymorphism leads to assortive mating, sympatric speciation can result
        • example: North American apple maggot fly, Rhagoletis pomonella
          • used to lay eggs only on hawthorn fruits, the food of its larvae
          • now one population also infests apple trees, the food of its larvae
          • because the fruits ripen at different times, the adults emerge and mate at different times
          • thus, there are behavioral and temporal barriers between the gene pools
          • there are allelic frequency differences between the two populations
          • they are in the process of sympatric population

          Reproductive isolation of gene pools

          • Habitat isolation: populations live in different habitats and do not meet
          • Temporal isolation: mating or flowering occurs at different seasons or different times of day
          • Behavioral isolation: little or no attraction between males and females
          • Mechanical isolation: structural differences in genitalia or flowers prevent copulation or pollen transfer
          • Gametic isolation: female and male gametes fail to attract each other or are not viable

          Genetic isolation of gene pools:

          • Reduced hybrid viability: hybrid zygotes fail to develop or fail to reach maturity
          • Reduced hybrid fertility: hybrids fail to produce functional gametes
          • Hybrid breakdown: offspring of hybrids have reduced viability or fertility

          6. Polyploidy can contribute to speciation.

          • polyploidy is a form of sympatric speciation that does not require geographic isolation
          • polyploidy means having more than two sets of homologous chromosomes
          • polyploidy occurs most commonly in plants as a result of errors during meiosis
          • the formation of even a single polyploid individual, if fertile, could be a speciation event if the plant reproduces:
            • asexually (vegetatively)
            • by self-fertilization

            Application: Speciation in the genus Allium by polyploidy.

            7. Allopatric leads to speciation through geographic isolation while sympatric leads to speciation through ecological isolation.

            Allopatric speciation occurs as a result of geographic isolation:

            • When two populations of a species are segregated by a geographic barrier such that they cannot interbreed, they are considered allopatric.
            • Dispersal mechanisms cause some individuals of a population to migrate to new locations, separating the parental, sympatric population, into two or more allopatric populations
            • allopatric populations that are reproductively isolated will diverge due to:
              • differing natural selection pressures due to slightly differing environments
              • differing mutation pressures due to the random nature of mutation
              • genetic drift, as chance is likely to produce slightly different allelic frequencies in the allopatric populations, especially if the number of founding members of the population is few.

              Sympatric speciation usually requires ecological/niche isolation:

              • Sympatric populations overlap in geographic distribution
              • a portion of a population may become fixed on resources not used by the parent population
              • differences in resource use can lead to a balance between two different adaptations, a balanced polymorphism
              • if the balanced polymorphism leads to assortive mating, sympatric speciation can result

              Any type of speciation requires reproductive isolation of gene pools

              • Many types of reproductive isolating mechanisms accelerate divergence between two populations undergoing speciation
                • Habitat isolation: populations live in different habitats and do not meet
                • Temporal isolation: mating or flowering occurs at different seasons or different times of day
                • Behavioral isolation: little or no attraction between males and females
                • Mechanical isolation: structural differences in genitalia or flowers prevent copulation or pollen transfer
                • Gametic isolation: female and male gametes fail to attract each other or are not viable
                • Reduced hybrid viability: hybrid zygotes fail to develop or fail to reach maturity
                • Reduced hybrid fertility: hybrids fail to produce functional gametes
                • Hybrid breakdown: offspring of hybrids have reduced viability or fertility

                8. Speciation due to divergence of isolated populations can be gradual.

                • Species descended from a common ancestor gradually diverge more and more in morphology
                • as they acquire unique adaptations
                • through the slow but relentless effects of natural selection.

                9. Speciation can occur abruptly.

                Punctuated equilibrium implies long periods without appreciable change and short periods of rapid evolution. :


                Future Prospects

                As sequencing technologies advance, computational tools will need to evolve in parallel to solve new technical challenges and support novel applications. For example, as the ability of sequencing platforms to produce longer reads becomes a reality, new mapping methods are required to accurately and efficiently align long reads. Because longer reads can span multiple exon𠄾xon junctions, the identification and quantification of alternative isoforms will improve significantly with the extra information encoded in longer reads. Furthermore, as laboratory methods mature to enable sequencing of minute quantities of RNA, complex statistical approaches will be needed to discriminate between technical noise and meaningful biological variation. These progresses will facilitate the analysis of transcriptomes in rare cell types and cell states, enabling researchers to reconstruct biological networks active at the cellular level. In addition, these advancements will allow transcriptome analysis to move into the field of clinical diagnostics for example, earlier monitoring of cancer screening and pregnancy could be accomplished by sequencing cancerous RNA or fetal RNA in the maternal blood. Furthermore, the integration of whole-genome sequencing with RNA-Seq in larger samples will provide greater insight into genetic regulatory variation. These experimental and bioinformatic advances will provide a powerful toolbox for fully characterizing the transcriptome as it relates to basic biological questions, as well as its rising impact on personalized medicine.


                3.E: Isolating and Analyzing Genes (Exercises) - Biology

                We are pleased to offer a series of self-paced tutorials on population genetic analysis that employ hand calculations and exercises within GenAlEx. These are drawn in part from the graduate workshops that we have offered (jointly and independently), around the world. Click the links below to download any tutorials of interest. In 2012, Tutorials 1-6 were revised to bring them up to date with the new features of GenAlEx 6.5. The newer Trouble Shooting Tutorial is strongly recommended for all users. It provides helpful tips for solving some of the issues that may prevent some data sets from running.

                An Introduction to Frequency-Based Population Genetic Analysis: scoring genetic markers, Allele Frequency, Heterozygosity, F-statistics, Nei Genetic Distance, Shannon Diversity Indices and Chi-square tests for Hardy-Weinberg Equilibrium

                Genetic Distance and AMOVA: Haploid, Codominant and Binary Genetic Distance, AMOVA and F-statistics

                Spatial Genetic Analysis: Principal Coordinate Analysis (PCoA), Mantel Tests for Matrix Correspondence and Spatial Autocorrelation Analysis

                Advanced Frequency-Based Analysis: DNA Profile Probability, Probability of Identity, Probability of Exclusion, Population Assignment and Pairwise Relatedness

                Advanced Features Including Data Import and Export: Working with DNA sequences, importing and processing raw genotypic data, exporting data from GenAlEx to other software. The Stats menu and how to customise the GenAlEx menu are also covered briefly.

                TwoGener: Male gametic inference, male gametic distances, gametic AMOVA

                Hierarchical Shannon Diversity Analysis

                This tutorial provides helpful tips for troubleshooting when GenAlEx is initially unable to run some data sets.


                T: 514-398-5634 | jean-benoit.charron [at] mcgill.ca (Email) | Raymond Building, R2-022a

                Degrees

                BSc Biochemistry (Université de Montréal)
                MSc, PhD Biology (Université du Québec à Montréal)

                Active Affiliations

                Senior Director of the Société canadienne de biologie végétale/Canadian Society of Plant Biologists
                Editorial Board Member Scientific Reports, Nature Publishing Group

                Research Interests

                Our lab studies the chromatin regulatory mechanisms that control stress tolerance in plants. Chromatin is a highly organized structure that can be modified in order to modulate the expression of genes. This control can be enacted via DNA methylation and/or through various types of histone modifications and nucleosome remodelling. These modifications can affect gene expression by changing the local chromatin state from either an “open” (transcriptionally active) or “closed” (transcriptionally repressed) configuration and vice versa. To control this process, specialized proteins will craft different combinations of modifications in order to activate or repress gene expression when necessary. The long term goal of our research is to understand how these specialized proteins modulate the dynamic tuning of the plant’s chromatin structure upon perception of environmental stress conditions, and how this translates the stress signals from the cellular environment into orchestrated responses from the DNA.

                To study chromatin dynamics we are using the monocot Brachypodium distachyon (purple false brome) as a genetic model system. This plant is a close relative of wheat and barley and is appealing for molecular studies due primarily to its small genome and the relative ease in which it can be transformed. The latter is crucial to our work in the lab as transgenic plants allow us to isolate and analyze the function of specialized chromatin modifying genes involved in the stress response mechanism. Development of over-expression and RNAi-mediated knock-down Brachypodium lines is currently underway in the lab. We employ a number of state-of-the-art molecular techniques (including RT-qPCR, ChIP-qPCR, ChIP-seq and RNA-seq) to identify and functionally characterize these chromatin modifying genes involved in stress response mechanisms in plants. We hope that the elucidation of the chromatin mechanisms involved in stress tolerance will ultimately lead to strategies for the improvement of important cereal monocot crops.

                In addition to further developing Brachypodium as a model organism we are also currently working with industrial hemp (Cannabis sativa), a dicot crop of particular economic importance in Canada. The molecular aspects of the stress response mechanisms of this crop are currently being analyzed in the lab. Furthermore, in collaboration with Agriculture and Agrifood Canada and Phytodata inc. we are developing accurate molecular assays for the simultaneous detection and quantification of resistant and sensitive isolates from airborne field fungus samples. These assays are needed for the development of environmentally acceptable crop protection strategies centered on minimum fungicide use.

                Current Research

                • Understanding the role of chromatin modifying complexes in the abiotic stress responses of cereal plant, Natural sciences and engineering research council of Canada (NSERC), Discovery grant
                • Improving yield and quality of Québec grown industrial hemp, Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (MAPAQ), Program to support innovation in the agri-food industry
                • Development of integrated management model of fungicide resistance, Ministère de l'agriculture, des pêcheries et de l'alimentation du Québec (MAPAQ), Innov’action Program
                • Infrastructure for a chromatin biology and epigenetics laboratory, Canada foundation for innovation (CFI), Infrastructure operating grant
                • Centre de recherche en sciences du végétal (Centre SEVE), Fonds de recherche du Québec – Nature et technologies (FRQNT), Strategic clusters

                Courses

                Life Sciences: The course integrates classical, molecular and population genetics of animals, plants, bacteria and viruses. The aim is to understand the flow of genetic information within a cell, within families and in populations. Emphasis will be placed on problem solving based learning. The laboratory exercises will emphasize the interpretation of genetic experimental data.

                • Restriction: Not open to students who have taken BIOL 202.
                • Terms
                  • Fall 2021
                  • Jean-Benoit Charron

                  Biotechnology: Practical laboratory-based research experience. Techniques in cellular and molecular biology, designing experiments and developing skills in interpretation and communication of experimental results.


                  Alkaline Lysis:

                  Alkaline lysis is a method used in molecular biology , to isolate plasmid DNA or other cell components such as proteins by breaking the cells open. Bacteria containing the plasmid of interest is first grown, and then allowed to lyse with an alkaline lysis buffer consisting of a detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. The detergent cleaves the phospholipid bilayer of membrane and the alkali denatures the proteins which are involved in maintaining the structure of the cell membrane. Through a series of steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed and the plasmid is isolated and purified.


                  3.E: Isolating and Analyzing Genes (Exercises) - Biology

                  In the exercise below you will be given an unknown DNA sequence and asked to use a web tool to translate the sequence into an amino acid sequence and hopefully identify the proper reading frame. You will then save this amino acid sequence to a word processing program (or e-mail it to yourself) if you want to use it in the next exercise.

                  Obtaining your sequence
                  In the lab, this might be obtained by sequencing a clone from a cDNA library or by isolating an amplified DNA fragment from a PCR amplification. Often, when we sequence such a product we find we have an unexpected fragment of DNA which we need to analyze. Here we will provide a partial sequence at random from our database of sequences. A partial nucleotide sequence will appear in the window below after you click on the Get Gene Sequence button.

                  Translating the Sequence
                  Several sites on the web perform a translation of an input sequence. Clicking on the Expasy link below will open a new window giving you access to a translation tool. Translating the DNA sequence is done by reading the nucleotide sequence three bases at a time and then looking at a table of the genetic code to arrive at an amino acid sequence. This program examines the input sequence in all six possible frames (i.e. reading the sequence from 5' to 3' and from 3' to 5' starting with nt 1, nt 2 and nt 3). What we typically look for in identifying the proper translation is the frame that gives the longest amino acid sequence before a stop codon is encountered. (Since there are 64 codons and three code for nonsense, we expect a stop codon to appear on average once every 20 amino acids if we simply read a sequence "out of frame". However, "on average" is just that, and it is possible to have an incorrect reading frame give an extended sequence with no stop codons. The next exercise will address that problem.

                  We will use Expasy tools for translation. Clicking on it will open a new window so you can return to this window for instructions and to copy your sequence.


                  Watch the video: AP Biology: Restriction Enzyme Digests on Circular Plasmids (July 2022).


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