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  Arthur Bergen: BIOT: September 1997  

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Subject: BIOT: September 1997
From: Arthur Bergen <a.bergen@ioi.knaw.nl>
Date: Mon, 6 Oct 1997 08:42:13 MET
Organization: ioi.knaw.nl
Priority: normal

New BIOTs!

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Edward Wilcox
Arthur Bergen
(BIOT editors)

This BIOT message contains:

        1) Do you know of any evidence for a GAPDH pseudogene
        2) A request for help in making a cDNA library from 100-5000 cells
        3) How do aberrant RNA splicing errors or PCR artifacts come about
        4) How can short tandem repeat loci from selected Philippine linguistic
           groups be used to compute genetic distances.
        5) The latest international ImMunoGeneTics database is now available
        6) A request for probes from the Retinoblastoma gene
        7) A request for a clone expressing the human EPO (Erythropoietin) gene
        8) By what mechanisms do pseudogenes arise

GAPDH pseudogene?                      9/3/97

        We are performing RT-PCR on some human tissue samples and have made
primers to GAPDH as a positive control. These primers are from the first
and fifth exons, therefore they should generate about a 320 BP band when
amplifying fully processed mRNA (cDNA) or a 2.2 Kb band when amplifying
genomic DNA. However, when we perform PCR on human genomic DNA we obtain a
320 BP band. We believe we have eliminated the possibility that the DNA is
contaminated  by RNA or the possibility that the PCR reaction (reagents) is
contaminated. At this time we believe the only other explanation is that
the human genome has a pseudogene for GAPDH. We have checked the Genbank
databases and find no evidence for a GAPDH pseudogene.

        Has anyone else ever experienced this same (or similar) problem? Is
there any evidence for a GAPDH pseudogene?

Thank you for any comments/suggestions.

Michael J. Kern, Ph.D.
E-mail: kernmj@musc.edu
        I am interested in making a cDNA library starting with material from
100-5000 cells only. Any help from people working with small number of
cells about methods/Kits for RNA extraction, RT-PCR, and cDNA library
construction is appreciated.

Mauricio Neira, Ph.D.
Department of Medicine
University of Wisconsin, Madison
Dear Sir/Madam,

        My project centers around developing the protein truncation test to
screen the ATM gene in familial breast cancer patients that are analyzed by
my department.
        I have noticed within my research the existence of aberrant RNA
splicing and am wondering how such splicing errors are caused. Such
splicing errors have been observed by other ATM researchers. I sequenced
one such fragment around 900 BP long. I was trying to generate the entire
ATM transcript of 9.2 kb but got this band instead. Upon sequencing, the
band contained 350 BP 5' end, 323 BP 3' end with the middle of this product
being intronic regions. Splicing did not occur at exon intron sites. Any
comments as to the nature of such aberrant splicing or PCR artifacts would
be greatly appreciated.

Yours faithfully,
Aaron Simpson
Clinical Chemistry
Western Australia
61 08 93408482
ph 08 9450 2197
fax 089 6455 225

        I intend to study genetic variation at short tandem repeat loci
among selected Philippine linguistic groups and compute for genetic
distances between them for my dissertation. This study shall be
contributory to our national effort of constructing a population database
primarily for forensic purposes and hopefully for other population studies.
My questions (among hundreds) include the following:

1.  What factors should I consider in choosing a "sample size" that would
give me the best estimate of actual population allele frequencies?

2.  At the minimum, how many loci/alleles do I need to study to allow for
computation of genetic distance between the linguistic groups (assuming
high frequency of heterozygotes for these loci)?

3.  What are the criteria for choosing a measure of genetic distance e.g.
Nei's,  and method of construction of a dendrogram e.g. Neighbor-joining or
UPGMA to best examine the phylogenetic relationships of these linguistic

As I've said, I have more to ask but I do not want to take so much of your
precious time (for now ;-)). Any input shall be greatly appreciated.

Thank you.
Jiji Miranda
Molecular Biology and Biotechnology Program
College of Science
University of the Philippines
Diliman, Quezon City
Dear Colleague,

I am pleased to send you the latest IMGT news for information and diffusion.
With many thanks.
Best regards.
Professor Marie-Paule LEFRANC

IMGT NEWS-August 1997

"Alleles and mutations"

IMGT, the international ImMunoGeneTics database, announces a STANDARDIZED
description of allele polymorphisms and mutations for all immunoglobulin
and T cell receptor V-REGIONs of all species, based on the IMGT unique
numbering (IMGT NEWS - March 1997). Allele alignments and tables for the
human IGH, IGK and IGL V-REGIONs are freely available at IMGT

IMGT initiator and coordinator :
Prof. Marie-Paule Lefranc
Laboratoire d'ImmunoGenetique Moleculaire, LIGM
UMR 5535 (CNRS - Universite Montpellier II)
1919 route de Mende
34293 Montpellier Cedex 5 - France
Tel: +33 (0)4 67 61 36 34 - Fax: +33 (0)4 67 04 02 31

IMGT reference : Giudicelli et al., Nucleic Acids Research, 25, 206-211(1997)
        I am working on the Retinoblastoma gene and we are in need of
probes for this gene.  Probes must be from within the gene. Sequences are
all we need, for we can make them ourselves.

Thank you very much.
Helizna Kilian
Department of Haematology
South Africa
email: GNHMHSK@med.UOVS.ac.za
Dear colleagues,

        I am looking for someone from whom I could obtain a pro- or
eukaryotic clone expressing human EPO (Erythropoietin) for in vitro

Thank you in advance, sincerely

Stefan Schmidt
        We have cloned and sequenced a "gene" of interest from a human
liver library. On analysis of the sequence, we believe that the "gene" is,
in fact, a pseudogene of a class of genes that are of interest to us. Can
anyone direct me to a recent review or book that deals with pseudogenes?
For example, we'd like to know the different mechanism by which pseudogenes
arise, their predicted abundance, importance, etc.

Thanks for any help.
Bill McIntire
Dr. Arthur A.B. Bergen
Department of Ophthalmogenetics
The Netherlands Ophthalmic Research Institute (IOI)
Royal Academy of Sciences of the Netherlands (KNAW)

** Snail-mail: **           ** FAX: **             ** E-mail: **

P.O.Box 12141               (+31)206916521         A.Bergen@IOI.KNAW.NL
1100 AC  Amsterdam
The Netherlands

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