UBC Research

Here is the proceedure used to generate the list of most of the genes that we isolated in the most recent projects. There are several more, but we ran out of time (and technology) to get them finalized and published. Cells under a low power microscope, at a low density. Just right for genetic study!

The above image shows a scattering of cells growing in a plastic flask which were started the day prior. At this stage of incomplete coverage, I changed their liquid environment to one that was missing magnesium ions. This low magnesium environment caused the cells to switch on many genes involved in magnesium collection, retention, and internal distribution. This genetic activation process involved reading the DNA to make messenger RNA, which is in turn read to make new proteins. It can take a few hours for this process to get fully involved, so I waited until then to collect the messenger RNA from these cells.

This low magnesium environment messenger RNA was then compared to another group of normal cell's messenger RNA using special grids of pre-made known messenger RNA sequences that would hold onto any similar sequence messenger RNA. The grid's beneficial feature is that it could give a useful estimate of the relative amount of any particular messenger RNA in the sample. The next step is to compare the normal and low magnesium samples to find any messenger RNA that had incresed in the low magnesium condition. These were then purchased and multiple copies made by growing them in yeast cells.
I then took these yeast cell messenger RNA copies and injected them into non-fertilized frog eggs, which after a few days of happily reading these messenger RNA copies as their own, would hopefully produce a functioning protein from them. A protein which we already knew to be involved in magnesium handling.

The last step was to use sensitive voltage and amperage measuring glass needle electrodes (shown above) poked just inside the frog egg to see if there was an increased permissiveness to magnesium entry into the frog egg through these new proteins. An increased movement of magnesium would show as a reduced resistance to the positive charged magnesium ions, or conversely, an increase in the electrical power (amperage) required to hold the frog egg's internal voltage constant while the outside magnesium varied.

Using the above computer controlled equipment, the process was fairly straight forward, I would step through a series of forced internal voltages in the frog egg, and see how much outward electrical current it took to balance the magnesium ion's inward movement at various concentrations.

This is the results from a control egg.
On the left: data collected when there was no magnesium on the outside. In the center: now with magnesium on the outside. On the right: the difference.
I can subtract the zero magnesium data from the magnesium data, and the result is pretty much flat data. Nothing happening here.

This is the results in the same order when testing an egg which has had messenger RNA injected. Now there are very definate results shown on the right.

This process also showed whether other ions besides magnesium were handled by the protein in question. The end result turned out to be a unique set ion transport parameters for each of the proteins tested. We were looking initially for a highly magnesium selective protein, however most of the proteins seemed to also handle a range of other ions. The important point however is that the other ions were rarely preferred over magnesium by the cell, and their naturally occuring concentrations would be so low that the most likely function of the protein was still magnesium handling.

So of the approximate 40,000 human genes, we can claim "function discovery" of about a dozen of them.
All of this information is collected with free public access on the National Center for Biotechnology Information (NCBI) website.

Metal transporter: CNNM2 (Oct 24, 2011)

Magnesium Transporter 1: MagT1 (Oct 24, 2011)

NIPA 1 (Oct 24, 2011)

NIPA 2 (Oct 24, 2011)

NIPA 3 (Oct 24, 2011)

Palmitoyltransferase ZDHHC17 :GODZ (Oct 24, 2011)

HIP14 (Oct 24, 2011)

Membrane magnesium transporter 1: MMgT1 (Oct 24, 2011)

Membrane magnesium transporter 2: MMgT2 (Oct 24, 2011)

Solute carrier family 41, member 1: SLC41A1 (Oct 24, 2011)

Solute carrier family 41, member 2: SLC41A2 (Oct 24, 2011)

Summary article: Ancient and modern magnesium transporters (Oct 24, 2011)
In the above summary article, we included some of our unpublished data
as a way to publish it. This includes the following:

N33, now called TUSC3 (Oct 24, 2011)

NIPA4, also known as Ichthyin (Oct 24, 2011)

SLC41A3 (Oct 24, 2011)

MagC (Oct 24, 2011)

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