Nutrition and Gene Expression

Lecture, Feb 12, 2015

Lipid Metabolism: Changesin Gene Expression

and Application to Studiesin Guinea Pigs and Humans

MEASUREMENT OF SPECIFIC mRNA LEVELS:

The most important method in study of gene expression.

We will discuss RT-PCR in detail, because of its widespread use.

R

MAKE THE INITIAL RNA

PROCESS TO mRNA:this is measured

TRANSLATE TO PROTEIN

MODIFY THE PROTEIN

NOTE: other steps can also bemeasured, if you want

The mechanism of how a gene is switched on isvery complicated and important. It usually requiresspecial proteins that bind to the REGULATORY ELEMENTof the gene. These proteins are usually calledTRANSCRIPTION FACTORS. We will study these proteinsin detail, for the March and April lectures continue discussof these proteins throughout the semester.

If there in an increase in the mRNA for a protein, youknow the synthesis of that protein has been increased.

Lots of research just looks at that: increase in mRNA.The newer methods are very interesting and powerful.

Reverse transcriptase-PCR (RT-PCR) is now the method of choice for measuring levels of specific mRNA.

Northern blots are an older, well-established method formeasurement of the amount of specific mRNA.

-mRNA for LDL receptor-mRNA for Cytochrome P7

Since RT-PCR is now extremely common, we will reviewthat method in detail.

Polymerase Chain Reaction (PCR):

TO MEASURE AMOUNTOF ONE SPECIFIC mRNA

IN A CELL SAMPLE

CELL#1: Few copies of

mRNA for acertain protein

CELL#2: Many copies of

mRNA for that protein

LYSE CELLSWITH INHIBITORS:mRNA ISOLATED

INTACT

mRNA:Sample #1

mRNA:Sample #2

NOTE: Many otherkinds of mRNAare also present!

The famous enzyme REVERSE TRANSCRIPTASE

is used to make a DNA copy of an RNA sequence.

Why is that called reverse transcriptase?

mRNA

strand

UUUCGAACCGGUCGUAUUCCGGG

AAAGCTTGGCCAGCATAAGGCCC

Reversetranscriptase

ComplentaryDNA strand

In this procedure, mRNA is REVERSE TRANSCRIBED back into a matching DNA strand (called: cDNA).

With reverse transcriptase, non-specific primersare often used to get cDNA for all the different kinds of mRNA in the cell.

Specificity is achieved when we amplify onlyone of the cDNA molecules.

You now have the COMPLEMENTARY DNASTRAND TO YOUR INITIAL mRNA

AAAGCTTGGCCAGCATAAGGCCC

With short primers SPECIFIC for that cDNA, you can get this replicated by a DNA polymerase.

AAAGCTTGGCCAGCATAAGGCCC

TTTCGAA

AAAGCTTGGCCAGCATAAGGCCC

TTTCGAACCGGTCGTATTCCGGGInitial product

of PCR reaction

AAAGCTTGGCCAGCATAAGGCCC

TTTCGAA

AAAGCTTGGCCAGCATAAGGCCC

TTTCGAACCGGTCGTATTCCGGG

Need abundant dATP, dCTP, dTTP, dGTPto provide bases for the new strand

Activity of DNA polymerase

The DNA polymerase has made a new DNA sequence, that is complementary to your original sequence

SPECIFIC PRIMER!

Once you have a 2d DNA sequence, you will need a primerto copy that sequence also.

RNA AND DNA, key differences:-uracil on RNA, thymine on DNA-ribose on DNA lacks OH group on ring

This OH onlypresent on theribose in RNA

No OH onat this spoton DNA

Double-strandedDNA

Heat to90C

Strands will separateat high temperature

Cool to65C

DNA polymerase makes a new complementary

sequence to each strand

REPEAT

Heat Cool

Each cycle: -heat to separate the strands

-cool and allow strand replication

Doubles the amount of DNA

After about 40 cycles, there is enough DNA to visualizewith simple ethidium bromide fluorescence on a gel.

Newer methods (Sybr Green, etc) measure the formationof the product right within the PCR machine. We do thatnow in Weed Hall and other labs (called real-time PCR).

The amount of cDNA that is made after manycycles (typically, 40) is roughly proportionalto the amount of mRNA isolated from thecells at the start of the experiment.

The cDNA sample is run on a gel, stained withethidium bromide, and quantified witha fluorescence gel reader.

WHICH BAND SHOWSMORE STAINING?

Sample1 Sample2

Gelmigration

This study in guinea pigs uses a food rich in stanols to lower plasma cholesterol.

The mechanism is complex, and involves recirculation of bile acids. This interesting study, which used both plasma and liver samples, showed some important effects on expression of genes in the liver involved in the biochemistry of lipids.

Corn Fiber Oil Lowers Plasma Cholesterol by Altering Hepatic Cholesterol Metabolism and Up-Regulating LDL Receptors in Guinea Pigs

Tripurasundari Ramjiganesh, Suheeta Roy, Hedley C. Freake, Jonathan C. McIntyre* and Maria Luz Fernandez

BILE SALTS ARE NEEDED FOR DIGESTION OF FATS. WE MAKE THEM IN THE LIVER FROM CHOLESTEROL.

SOME BILE SALT IS USUALLY REABSORBED AND USED AGAIN.What would happen if the bile salts were NOT reasorbed?

The liver uses cholesterol (largely imported from the plasmathrough the LDL receptor) to make bile acids needed for lipid digestion in the small intestine.

MUCH OF THIS CHOLESTEROL IS OBTAINED BY GETTINGCHOLESTEROL-RICH LDL FROM THE PLASMA.

THE LDL ARE IMPORTED WITH THE LDL RECEPTOR ONTHE SURFACE OF THE HEPATOCYTE.

These compounds in corn fiber oil (called STANOLS)can BLOCK the re-asorption of bile acids.

This has striking effects on metabolic events in the liver!The liver has to make MORE BILE ACIDS..using cholesterol from some source. LDL is a very good source.

LIPOPROTEINS CONSISTLARGELY OF CHOLESTEROL

The upper panels show intensity of mRNA for the

LDL receptor in liver, which increases with either

corn fiber oil, or with low cholesterol diet.

DISCUSSION: How has corn fiber oil changed the biochemistryof the guinea pig?

-Which individual metabolic pathways are now altered?

-How could we determine if this were happening in a person who added this to their diet?

WHAT KIND OF CHANGES IN GENE EXPRESSION

CAN BE TYPICALLY STUDIED IN HUMANS?

What questions were asked in the study of humans

placed on a weight-reduction program?

NOTE: Changes in biochemistry and gene expression

in the liver are a MAJOR topic in nutrition..but we

usually can’t measure that directly by getting

human liver biopsies!

What CAN we do instead? Important question.

STUDY USING CIRCULATING WBC IN HUMANS:

The Lowering of Plasma Lipids following a Weight Reduction Program Is Related to Increased Expression of the LDL Receptor and Lipoprotein Lipase

Madhu Patalay, Ingrid E. Lofgren, Hedley C. Freake, Sung I. Koo, and Maria Luz Fernandez

Journal of Nutrition, 135: 735-739, 2004

LDL-receptor gene, chromosome 19,at P13.3

Lipoprotein lipase geneChromosome 8, at P22

Lipoprotein lipase (LPL) acts to convert trigylcerides to glycerol and free fatty absorbs, which are then transported into the cell.

Mononuclear cell isolation. Mononuclear cells were isolated from whole blood through centrifugation on a Ficoll gradient according to the method of Boyum (21).

NOTE: Mononuclear cells are a class of white cells. They are usually 3-5% of the totalWBC, and upon stimulation can become active macrophages. Many diet studies lookat this class of WBC, because they are thought to play a role in heart disease.

DATA FROM TWO SUBJECTS IN THE STUDY: Shows changes in specific mRNA levels for several genes.

RT-PCR USEDFOR THIS

MEASUREMENT

DISCUSSION:

-Which CLASS of proteins changed in monocytes of subjectson a weight-loss program?

-What can we conclude about metabolites available to monocytes,which are active cells that absorb many components fromthe bloodstream?