Notes on gene amplification Biol.G4054y

Historically: John Littlefield (Harvard, then Hopkins): searching for gene regulatory mutations in cultured cells

dhfr - metabolic role - folic acid vitamin - FH4

for purine nucleotide and thymidylate synthesis - DNA synthesis

methotrexate (amethopterin): looks like FH₂ substrate, fools enzyme, ties up enzyme

used for anti-cancer chemotherapy via inhibition of DNA synthesis

a purine and thymidine requirement induced in presence of MTX (HAT medium).

MTX^R mutants - operational selection: MTX -thy ® colonies at 10^-5.

= MTX-resistant. Why resistant?

(Littlefield wanted gene activity-up mutations, to learn about control of gene activity.)

But: perhaps mutation changed dhfr enzyme so no longer fooled by MTX

Found MTX-sensitivity of mutant enzyme = sensitivity of normal, or "wild-type". OK.

Regulatory mutation would result in amount of enzyme up. Measure amount of catalytic activity: up 10x. OK.

But: could be a more active mutant enzyme.

So: measure actual protein directly. Had to separate dhfr protein (0.01%) from rest of cell protein. Measured radioactivity in immunoprecipitates or after electrophoretic separation (SDS gels) after labeling cells with a radioactive amino acid

Found dhfr protein up. OK.

Maybe some new factor makes translation of dhfr mRNA ® protein more efficient in mutant.

So: isolated total mRNA, translated it in a test tube, using protein translation machinery from red blood cells. Mutant ® 10X more dhfr protein here, just as in mutant cell

OK. dhfr mRNA level up, as measured by mRNA activity

So either gene is more active in turning out mRNA, or ...perhaps more genes, of normal activity???

Meanwhile, June Biedler (Sloan-Kettering): finding that MTX-resistant hamster cells contain chromosome abnormalities

HSRs (homogeneously staining regions, compared to the usual banded appearance)

Usually on the same chromosome

Gradually lost (1-2 year period), along with MTX-resistance.

In this (just) pre-recombinant DNA era, then:

Bob Schimke (Stanford): purified the dhfr mRNA from a MTX^R mutant (mouse cell line) where it was present in high levels (100X), radioactively labeled it, used it as a probe via solution nucleic acid hybridization. Tour-de-force purification (from 1%) by a young grad student, Fred Alt, by subtractive hybridization (one of the first applications of this strategy):

‑ Resistant cell mRNA ® 3H‑cDNA.

‑ High cot hybridization to excess of WT mRNA: isolate unhybridized (single stranded) cDNA with hydroxyapatite

‑ Low cot hybridization to resistant cell mRNA: isolate hybridized (double‑stranded) cDNA with hydroxyapatite.

Use this gene-specific probe to quantify DNA per cell in normal vs. mutant cells:

Had done all the Littlefield experiments on this mouse cell line:

MTX^R mutant had 100X more enzyme, 100X more translatable mRNA and, now find: 100X more dhfr DNA. That is, 100X more dhfr genes.

Finally, what is the relationship of these extra genes to the HSRs (which were 100-300 times too large to be just the extra genes).

Do the high levels of dhfr trigger a specific chromosomal abnormality, or is the HSR actually represent the site of dhfr gene amplification?

Jack Nunberg in the Shimke lab, with Gail Urlaub and L. Chasin (Columbia): in situ hybridization of this radioactive dhfr probe to metaphase chromosome spreads from an amplified Chinese hamster cell line: hybridized to the HSRs of the chromosomes:

Size of HSR = 30X size of the 200 dhfr genes present.

[5 pg DNA/cell, or 5 X 10^-12 g/cell ¸ 660 g/mole = 7.6 x 10^-15 moles, X 6 X 10²³ molecules/mole = 4.5 X 10⁹ base pairs/cells, or 4.5 X 10⁶ kb/cell. HSR = 3.5% of chromosome length, = 160,000 kb, ¸ 200 amplicons = 800 kb/amplicon. Dhfr gene is 25 kb, or only 3% of amplicon.]

So amplified region repeating unit ("amplicon") >>> than gene itself.

Nowadays: fluorescent probes.

Two chromosomal forms of amplified genes :

1- HSRs or ABRs (abnormally banded regions) , stable.

2- DMs, (double minutes) unstable

HSR DNA structure: tandem duplication

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Sometimes head-to-tail instead of head to head:

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Two basic ideas:

1. gene over-replication, replicon gone wild (onion‑skin). A replicon is a unit of DNA replication (replication eye).

- must be followed by some kind of recombinational joining to tie up loose ends.

- perhaps there is a transitory existence as a free, circular, amplicon

- (site of HSR or amplified genes may be close to but distinct from the orginal locus.)

2. unequal sister chromatid exchange.

Parallel slipped alignment - one chromatid with a deletion and one with a duplication

Antiparallel (doubled back) - Acentric fragment (lost) + fused chromatid that must break in mitosis, yielding two unequal arms, one with the target gene duplicated.

In situ hybridization analysis of early events in dhfr amplification support mechanism #2 (Hamlin, Trask).

Generality: Not just dhfr. Found almost whenever the selective conditions are right:

1. a good inhibitor of gene product, need for more of the gene product

2. a multistep selection, to allow a multistep process (CAD, TS, pyrimidine biosynthetic enzymes, multidrug-resistance)

This all in the lab. How about in nature?

In real normal nature:

Frog embryos: genes for ribosomal RNA, needed for protein synthesis, amplified and present as mini-circles in the developing egg (oocyte).

Fruit fly larvae: the gene for the chorion proteins that coat the egg are amplified in the cells that surround the egg (follicle cells) at a particular stage in development. In this case there are signals in the DNA surrounding the chorion genes that trigger the amplification.

But these are exceptional cases, not the usual mechanism by which a cell makes a lot of a gene product (i.e., via a high transcription rate)

Natural, but pathological: in the development of tumors themselves:

N-myc amplification in lung cancers and neuroblastomas. myc seems to have a function in cell-cycle control, or commitment to a round of DNA synthesis that is part of cell division. DMs abound in these cells. In lung cancer, myc amplification is associated with aggressive growth and a poor prognosis.

In medicine: cancer chemotherapy. MTX-resistance in a tumor can occur by gene amplification of dhfr (but not often documented).

More common: mdr1 gene (multi-drug resistance) ® membrane protein that pumps a variety of compounds (adriamycin, actinomycin, colcemid) out of the cell, including many of the anti-cancer drugs. Gene amplification ® overproduction of mdr1 ® drug resistance: tumor cells, previously killed by a drug, now resume growth.

In biotechnology industry:

To isolate a mammalian cell line producing large amounts of a useful protein:

Select amplificants for dhfr using MTX. Your favorite gene co‑amplifies along with dhfr

Join dhfr gene and EPO and transfect a cultured mammalian cell. Isolate a transfectant. Grow it in increasing amounts of MTX. Get a cell line that is highly resistant to MTX, has 1000 dhfr genes, and has, also, 1000 EPO genes. These cells will synthesize and secrete massive amounts of EPO, normally made in small amounts in the kidney. Manufacture EPO, inject it into people, cure anemia, kidney disease, cancer (facilitates re-population of blood-forming cells after aggressive radiation therapy).