Advanced Environmental Biotechnology II

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Advanced Environmental Biotechnology II

• Week 06 - expression analysis of functional genes

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• 5RT-PCR and mRNA expression analysis of
  functional genes
• Balbina Nogales

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• 5.1 Introduction
• Microbial communities are essential for the
  functioning of ecosystems.
• Their activity is regulated by environmental
  factors and depends on the activities of their
  individual members and populations and
  interactions amongst them.
• They are complex, metabolically flexible and
  highly adaptable to changing environmental
  conditions, with their function finely regulated
  at the molecular level.

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• The analysis of microbial processes in the
  environment an important challenge for microbial
  ecologists.
• Ecophysiological approaches rely on the detection
  of products resulting from a particular process
  and the measurement of transformation rates.
• Now molecular biology techniques, such as the
  analysis of gene expression via detection of mRNA
  after reverse transcription-polymerase chain
  reaction (RT-PCR), can show microbial function.

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• Reverse transcription occurs in the cytoplasm of
  cells infected by viruses. In this process, viral
  ssRNA is transcribed by the viral reverse
  transcriptase (RT) into double stranded DNA.
• Reverse transcription takes place in 3'?5'
  direction. tRNA ("cloverleaf") hybridizes to PBS
  and provides -OH group for initiation of reverse
  transcription.

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• 1) Strong stop complementary DNA (cDNA) is
  formed.
• 2) Template in RNADNA hybrid is degraded by
  RNase H domain of reverse transcriptase
• 3) DNAtRNA is transferred to the 3'-end of the
  template (synthesis "jumps").

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• 4) First strand synthesis takes place.
• 5) The rest of viral ssRNA is degraded by RNase
  H, except for PP site.
• 6) Synthesis of second strand of ssDNA is
  initiated from the 3'-end of the template. tRNA
  is necessary to synthesis of complementary PBS.

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• 7) tRNA is degraded
• 8) After another "jump", PBS from the second
  strand hybridizes with the complementary PBS on
  the first strand.
• 9) Synthesis of both strands is completed by the
  DNAP function of reverse transcriptase.

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• Both dsDNA ends have U3-R-U5 sequences, so called
  long terminal repeat sequences (3'LTR and 5'LTR,
  respectively). LTRs mediate integration of the
  retroviral DNA into another region of the host
  genome.

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• Since prokaryotic gene expression is a finely
  regulated process, detection of transcripts for a
  given gene constitutes significant evidence of
  the occurrence of a given biological process
  within the environment. Recent years have seen a
  significant increase in the number of studies
  reporting the analysis of microbial gene
  expression by RT-PCR in environmental systems.

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• Since prokaryotic gene expression is a finely
  regulated process, detection of transcripts for a
  given gene constitutes significant evidence of
  the occurrence of a given biological process
  within the environment. Recent years have seen a
  significant increase in the number of studies
  reporting the analysis of microbial gene
  expression by RT-PCR in environmental systems.

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• Most are in three main groups.
• (i) analysis of gene expression in pathogenic
  bacteria, e.g. Staphylococcus aureus and
  Helicobacter pylori
• (ii) detection and analysis of expression of
  genes involved in biogeochemical processes eg
  methanotrophy, nitrogen fixation, nitrification,
  denitrification and carbon fixation
• (iii) investigation of the expression of genes in
  the biodegradation of environmental pollutants,
  eg aromatic hydrocarbons.

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• 5.2 Advantages and limitations of the RT-PCR
  analysis of bacterial functional genes
• Several environmentally relevant questions can be
  approached by using RT-PCR-based techniques as
  shown in Figure 5.1.

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• Figure 5.1
• Schematic view of the different RT-PCR approaches
  that can be applied to gene expression analysis
  in environmental studies.

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• RT-PCR can be used to detect transcription of a
  gene of interest and to determine the diversity
  of the transcripts being expressed in the
  environment. The individual microorganisms in
  which specific gene expression is occurring can
  be identified by in situ RT-PCR techniques, and
  these organisms can be enumerated and their
  spatial distribution determined. Thirdly, the
  effect of environmental parameters in gene
  expression can be explored by quantitative RT-PCR
  and by using techniques for global gene
  expression analysis such as RNA fingerprinting by
  arbitrarily primed PCR (RAP-PCR) or differential
  display (DD), which allow for the analysis of
  modulation of gene expression in response to
  changing environmental conditions.

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• Techniques known in general as RNA fingerprinting
  include differential display and RNA
  fingerprinting by arbitrary primed PCR (RAP-PCR).
  Both methods are based on PCR amplification of
  random subsets of genes from two or more RNA
  samples. The first step of either is to
  reverse-transcribe random subsets of mRNA to
  cDNA.
• In differential display, this is done using an
  anchored primer, which is typically a polyT
  oligonucleotide with one or two additional bases
  (e.g., T12AC). In contrast, RAP-PCR uses
  arbitrary primers in reverse transcription. These
  primers are typically 10 bp in length and may
  anneal to complementary sequence and prime
  reverse transcription from any point along an RNA
  transcript.

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• Differential display.
• Total RNA is extracted from two experimental
  samples, 1 and 2. An anchored primer, such as
  T12G, is used to reverse-transcribe a subset of
  the mRNA to cDNA.
• Random fragments of the cDNA transcripts are
  amplified in duplicate PCR reactions using
  different combinations of forward and reverse
  primers.
• The resulting PCR products are compared to
  identify gene fragments expressed in sample 1 but
  not 2 (iii), expressed in 2 but not 1 (ii), and
  expressed in both 1 and 2 but at different
  intensities (I).

from D. E. Moody (2001) Genomics techniques An
overview of methods for the study of gene
expression J Anim Sci 2001. 79E128-E135.
jas.fass.org/cgi/reprint/79/E-Suppl/E128.pdf
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• RT-PCR methods are not intrusive (no incubation
  of samples, nor addition of substrates required).
  
• RT-PCR-based analysis of mRNA is also culture
  independent, sensitive, specific, rapid,
  reproducible and can be adapted to
  high-throughput systems when the analysis of many
  samples is required.

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• But the analysis of gene transcription by RT-PCR
  has important methodological limitations.
• Firstly, prior knowledge of the sequence of genes
  of interest is a prerequisite of such analysis to
  enable the design of primers to allow
  amplification of specific RT-PCR products.
• Some of the limitations of RT-PCR techniques are
  more methodological, such as the quantity,
  quality and stability of the RNA extract to be
  used in the reaction.
• RT-PCR amplification from environmental samples
  would be limited by difficulties in the RNA
  extraction and by the presence of inhibitory
  substances in the extracts which will interfere
  with the RT-PCR reaction.

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• Finally, since RT-PCR is highly sensitive,
  controls to ensure that amplification products
  are not derived from contaminating DNA need to be
  performed with every RNA extract used in RT-PCR
  reactions. Typically, RNA extracts are treated
  with RNase-free DNase and used in PCR reactions
  without a prior RT reaction (no-RT control).

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• 5.3 The RT-PCR reaction
• RT-PCR (19) consist of two sequential steps,
  namely a reverse transcription reaction (RT) in
  which a complementary DNA molecule (cDNA) is
  generated from an RNA template by extension of an
  oligonucleotide primer due to the action of a
  reverse transcriptase, and a subsequent PCR
  amplification reaction in which the cDNA is
  amplified exponentially by a thermostable DNA
  polymerase. Both reactions can be performed
  separately or sequentially in the same tube. Most
  suppliers of molecular biology products maintain
  excellent web pages with useful technical
  information on RT-PCR procedures (Table 5.1).

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• 5.3.1 The RNA template
• It is important to have high quality RNA, i.e.
  not degraded and free from DNA and ribonuclease
  contamination, or substances inhibitory to
  enzymatic reactions.
• Prokaryotic mRNA is a labile molecule with a
  short half-life.
• Considerable care is needed to avoid RNA
  degradation.
• RT-PCR amplification of prokaryotic mRNA is
  usually performed using total RNA extracts that
  also contain the more abundant RNA fractions
  ribosomal (rRNA) and transfer (tRNA) RNA.

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• 5.3.2 Primers for reverse transcription
• Three types of primers can be used
• (i) specific primers that anneal exclusively to
  the mRNA of interest
• (ii) random hexanucleotides that anneal randomly
  to any RNA molecule (including rRNA) present in
  the extract
• (iii) oligo (dT) primers that bind to poly(A)
  tails at the 3'-end of mRNAs (rarely used for
  prokaryotes).
• The objective is to obtain a high proportion of
  cDNAs complementary to the target RNA and with
  the maximum possible length.
• The choice would in most cases be the use of
  specific primers for the RT reaction.
• Random hexameric primers leads to the
  transcription of non-coding RNA fractions in
  addition to the mRNA fraction, although it offers
  advantages when the product of a single RT
  reaction is to be used in several PCR reactions
  using different primer sets.

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• 5.3.3 Reverse transcriptases
• There are two reverse transcriptases of viral
  origin, the avian myeloblastosis virus reverse
  transcriptase (AMV) and the Moloney murine
  leukaemia virus reverse transcriptase (M-MLV),
  and two of bacterial origin, C. therm polymerase
  (from Carboxydothermus hydrogenoformans) and Tth
  DNA polymerase (a recombinant DNA polymerase
  derived from Thermus thermophilus).
• Both bacterial reverse transcriptases are
  thermostable enzymes, thereby enabling the
  reaction to be performed at high temperatures and
  thus providing optimal conditions for primer
  annealing and reducing the effects of secondary
  structure in the template.

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• Bacterial reverse transcriptases have special
  features that have made them the polymerase of
  choice for certain applications. Tth DNA
  polymerase has the ability to perform both RT and
  PCR amplification in the presence of manganese or
  magnesium ions, respectively and forms the basis
  of the one-step one-enzyme RT-PCR protocols. C.
  therm polymerase is the only reverse
  transcriptase with an associated 3' to 5'
  proofreading activity, which results in an
  increased fidelity.

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• 5.3.4 One-step and two-step RT-PCR protocols
• one-step procedure (also called continuous)
• two-step procedure (or uncoupled)

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• In the one-step procedure, the RT and the PCR
  reactions take place sequentially in the same
  tube, in the presence of a unique buffer using a
  specific primer set, therefore no additional
  reagents need to be added during the reaction.
  One-step RT-PCR can be carried out using a single
  enzyme (Tth DNA polymerase), referred to as
  one-enzyme protocols, or by using commercially
  available combinations of reverse transcriptase
  plus thermostable DNA polymerase (two-enzyme
  one-step protocols). The one-step procedure
  requires less manipulation, reducing pipetting
  errors, time and minimizing the risk of
  contamination.

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• In the two-step procedure, RT and PCR reactions
  are done sequentially, but separately, in the
  optimal reaction conditions for each enzyme
  because a two-buffer system is used. The RT
  reaction can be performed with specific primers,
  random hexanucleotides or oligo (dT) (except when
  using Tth polymerase which requires the use of
  specific primers).
• After the RT reaction is completed, PCR reagents
  can be added to the tube (one-tube system) or
  an aliquot of the RT reaction is transferred to
  another tube and a PCR reaction with specific
  primers is performed (two-tube system).
• The two-step procedure, especially the two-tube
  system, provides greatest flexibility since the
  cDNA can be used in several PCR reactions using
  different primer sets. It does require more
  manipulation, increasing the likelihood of
  contamination.

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• Sensitivity seems to be greatest using the
  one-step two-enzyme RT-PCR protocol, followed by
  the two-step two-enzyme protocol with lowest
  sensitivities reported using the one-step
  one-enzyme (Tth polymerase) protocol (24).
  Factors other than sensitivity may also dictate
  the choice of RT-PCR protocol including
  requirements related to the product yield
  required, reaction specificity, or the number of
  samples to be processed.

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• 5.4 Quantitative RT-PCR
• Quantitative RT-PCR methods have been developed
  for the quantification of steady-state mRNA
  levels. Quantification can be performed after the
  reaction is completed (end-point measurement) or
  during the course of the reaction (real-time or
  kinetic).
• In some cases absolute quantification of the
  number of target copies per specific unit is
  performed, but most often quantitative RT-PCR is
  used to quantify changes in the expression of a
  specific gene after a treatment by comparison to
  a reference sample such as an untreated control
  (relative quantification).

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• The most frequently used quantitative RT-PCR are
  competitive RT-PCR and real-time RT-PCR. Both
  techniques are reproducible and have comparable
  sensitivity.
• Competitive RT-PCR is based on the
  co-amplification of the target RNA and known
  amounts of a standard RNA (the competitor) that
  is designed to be amplified with the same primers
  and with the same efficiency as the target but
  differs in length or contains a mutation that
  allows its differentiation from the target.
• Real-time RT-PCR, product synthesis is measured
  in each cycle during the exponential phase of PCR
  by measuring an increase in fluorescence as the
  reaction proceeds. Examples of quantitative
  analyses of gene expression in environmental
  samples are the quantification of
  ribulose-1,5-biphosphate carboxylase/oxygenase,
  rbcL, transcripts in diatom cultures and marine
  samples and the quantification of ammonia
  monooxygenase, amoA, transcripts in a biofilm.

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• 5.5 Analysis of global gene expression
• Genome-wide screening of mRNA transcripts,
  produced under different environmental
  conditions, can be compared in order to determine
  genes that are differentially expressed (induced
  or repressed) under varying conditions. Several
  methods are available for analyzing global gene
  expression in prokaryotes including differential
  display (DD) and RAP-PCR. The basic principle of
  DD and RAP-PCR is the generation of a collection
  of cDNAs using short oligonucleotides that in
  theory covers the whole genome. The cDNAs are
  subsequently amplified by PCR and the products
  separated on polyacrylamide or agarose gels.
  Comparison of the band patterns allows for the
  detection of differentially expressed genes.

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• Band patterns are checked by other techniques
  such as quantitative RT-PCR, as the frequency of
  false positives generated by DD or RAP-PCR may be
  high.
• The DD method uses anchored oligo (dT) primers
  for the RT reaction, followed by PCR
  amplification using the same anchored oligo (dT)
  primers and short arbitrary primers.
• RAP-PCR uses arbitrary primers only, both for the
  RT and PCR reaction, so has been used most to
  analyze prokaryotic mRNAs.

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• In studies of prokaryotic systems, several
  different strategies with respect to the type of
  primers used for DD and RAP-PCR have been used.
• Primers based on the calculation of oligomer
  frequency distribution in the coding regions of
  the genome of Enterobacteriaceae.
• Use of arbitrary primers together with a primer
  based on Shine-Dalgarno sequences from the 5'-end
  of bacterial mRNAs.
• To decrease the number of false positives caused
  by rRNA, use antisense probes for 16S and 23S
  rRNA, which show the fragments amplified from
  rRNA.

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• Another approach uses a large number of arbitrary
  primers to allow high-density sampling of
  differentially expressed genes.
• Designed to avoid annealing of the arbitrary
  primers to stable RNAs such as rRNA.
• RAP-PCR has analysed differential gene expression
  in prokaryotes, in particular for the analysis of
  genes involved in environmentally relevant
  processes such as response to pollutants,
  symbiotic associations and sulfate respiration.

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• Apart from DD and RAP-PCR there is a variety of
  related techniques relying on reverse
  transcription and PCR amplification procedures,
  including cDNA-amplified fragment length
  polymorphism (cDNA-AFLP), cDNA representational
  difference analysis (cDNA-RDA), cDNA-RNA
  subtractive hybridization and most recently DNA
  microarrays.

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• 5.6 Conclusions
• Questions such as which genes are expressed in
  the environment, how diverse the transcripts are,
  what are the transcription rates in the
  environment, how is gene expression affected by
  changing environmental conditions and which
  microbes are expressing the genes of interest can
  be approached by RT-PCR techniques.
• Results from RT-PCR studies should significantly
  contribute to our understanding of the
  functioning of microbial processes in the
  environment.
• A key challenge is still to obtain sufficient
  amounts of high quality RNA from the environment,
  and in particular from environments in which
  microbial biomass is limited and/or inhibitory
  substances (such as humic acids) are present.