HDTenEightyP/NetNews huggingface dataset:
From: bertheau@inapv.inapg.inra.fr (Yves Bertheau)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: 2nd round of PCR
Date: 19 Aug 92 07:46:13 GMT
Distribution: bionet
Organization: Institut National de la Recherche Agronomique INA P-G

In article <k?o.714144120@soma.umdnj.edu> byrne@soma.umdnj.edu (Bruce Byrne) writes:
>walterh@U.WASHINGTON.EDU (Walter Hill) writes:
>
>>Dear fellow net-rats:
>>A colleague of mine is amplifying rotavirus gene using RT-PCR and that works
>>well and a second round of PCR cycles using nested primers also works well.
>>However, when the second amplification is performed with primer identical to
>>the first amplifcation, no or very little amplification occurs.
>>Can anyone provide an explaination for this phenomenon?
>
>Maybe.  If the original target were rare or not a very good fit to your 
>primers, your colleague likely formed some primer dimers.  Those unwanted
>PCR products are perfect targets for the second round of amplification by
>the same primer pair.
>
>Added note of caution.  Any second round of amplification adds great
>complexity to handling carryover contamination.  
>
>Cheers!
>
>Bruce Byrne

We used a second amplification (25 cycles for the 1st one and the 2nd one) to
detect our phytopathogenic bacteria. We used only 5 ul of the 1st
amplification and it works. We never tested the recovery of all the DNA
of the 1st amplification...

No real idea why it does no work in your case...

Sincerely,
-- 
		Yves Bertheau
INRA INA P-G		Pathologie Vegetale	16 rue Claude Bernard	
75231 PARIS cedex 05	FRANCE	Tel +33 (1) 43.31.93.97 
Fax: +33 (1) 43.31.83.82 Internet: bertheau@inapg.inra.fr
From: adm@neuro.duke.edu (Alan Magid)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Lab stocks database: re-re-re-inventing the wheel
Summary: Funding sources for biosoftware
Date: 16 Aug 92 12:25:10 GMT
Distribution: na
Organization: Duke University; Durham, N.C.

The National Library of Medicine (branch of NIH) has a well-funded
grants program for BioTech software development. Considering the need
expressed on the net, a competitive (i.e. fundable) application to
support the software development could be put together by a qualified
software development group.
Alternatively, a private company (even a one-person shop) could get SBIR
money to do the development. The small business grants program of the
NIH, for example, will give upto $50,000 for a 6 month feasibility
study. Contact NIGMS I would guess. Ask your grants and contract office
for help with exact names and addresses.
Newsgroups: bionet.molbio.methds-reagnts
From: kim@m44.unm.edu
Subject: Lysate RNAase Protection Assay
Date: Wed, 12 Aug 92 18:13:33 GMT
Organization: UNM Cancer Center, Albuquerque, NM
Reply-To: kim@m44.unm.edu

Hello:

Has anyone tried out the "Lysate RNAase Protection Assay" of Haines and
Gillespie (BioTechniques June, 1992)?

Does it really work?  Is it comparable in sensitivity to RPA using purified
RNA?  Is there a problem with the fact that there seems to be no step for
denaturing the probe/target molecules by heating prior to hybridization?

I understand there is a kit available.  Is it substantially different from the
procedure described in the paper?

Thanks.

Daniel Kim
KIM@FLOVAX.LANL.GOV
From: walterh@U.WASHINGTON.EDU (Walter Hill)
Newsgroups: bionet.molbio.methds-reagnts
Subject: 2nd round of PCR
Date: 17 Aug 92 20:12:20 GMT
Distribution: bionet

Dear fellow net-rats:
A colleague of mine is amplifying rotavirus gene using RT-PCR and that works
well and a second round of PCR cycles using nested primers also works well.
However, when the second amplification is performed with primer identical to
the first amplifcation, no or very little amplification occurs.
Can anyone provide an explaination for this phenomenon?
Any guesses (well, almost any) and/or suggestions will be greatly appreciated.
Many htanks in advance, -Walt Hill

walterh@u.washington.edu (primary)           (secondary) WEH@FDACFSAN.BITNET
Food & Drug Admin., Seafood Products Research Center, Bothell, WA 98041-3012
VOICE:(206) 486-8788           "Cook it first"            (206) 483-4996:FAX
From: kofoid@bioscience.utah.edu (Eric C. Kofoid)
Newsgroups: bionet.molbio.methds-reagnts
Subject: RNA ligase & single-stranded DNA
Date: 14 Aug 92 17:04:15 GMT
Organization: Dept. Biology, University of Utah
X-Xxmessage-Id: <A6B1442F9301C399@ls-24.biology.utah.edu>
X-Xxdate: Fri, 14 Aug 92 18:06:23 GMT
X-Useragent: Nuntius v1.1.1d7

I am going ligate oligonucleotides into linearized single-stranded
plasmid 
DNA using RNA ligase. When I last did this (years ago), it was very 
inefficient. I would appreciate any favorite enhancements or references
to 
this method. 

 __________________________________________________________________
|  Eric Kofoid                         Dept.Biology, U. of Utah    |
|  "Being certain of the conclusion assists in finding the proof"  |
|                                      Galileo, 17th century       |
|__________________________________________________________________|
From: GBGA35@UK.AC.GLASGOW.VMS3
Newsgroups: bionet.molbio.methds-reagnts
Subject: PCR Questions!!
Date: 19 Aug 92 13:58:00 GMT
Distribution: bionet
Original-To: METHODS@UK.AC.DARESBURY

Hello Net People,

Apologies for the naivity of the following two questions; I'm not a molecular 
biologist ....yet. Answers will be gratefully received.

1. After asymetric PCR I would like to quantify the DNA present (typically in
c. 20ul). Any suggestions as to methods, references etc.?

2. Is it possible to extract mtDNA, for PCR, from samples that have been stored
in formalin or alcohol? Again are there any protocols or references?

Thanks very much,

Mark Willcox					Tel. 041-339-8855
Institute of Genetics				Fax. 041-330-5994
University of Glasgow				E-Mail:GBGA35@uk.ac.glasgow.vms
Church Street
Glasgow   G11 5JS
UK
From: penas@ucbeh.san.uc.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Screening cDNA Libraries
Date: 12 Aug 92 21:57:26 EST
Distribution: world
Organization: Univ. of Cincinnati

Hello!

I am soon going to start screening a lambda gt10 cDNA library.  This has never
been done successfully in my lab.  I would greatly appreciate any
recommendations for proper hybridization conditions.  We have a rotating
hybridization oven and perform all our hybridizations in hyb. bottles.

A million thanks in advance for any type of suggestions or usefule comments.

Sincerely,
-- 
Sandra Pena de Ortiz     : penas@ucbeh.bitnet
University of Cincinnati
College of Medicine
Toxicology Program
From: scott@phylo.life.uiuc.edu (Scott Howard)
Newsgroups: bionet.molbio.evolution
Subject: Re: Making Evolution More Efficient
Date: 19 Aug 92 13:02:15 GMT
Organization: University of Illinois at Urbana

Hard to speculate until you define what you mean by efficient-
Fewer cycles to an optimized subroutine?
From: una@phy.duke.edu (Una Smith)
Newsgroups: bionet.molbio.evolution
Subject: Re: Was There Once an RNA World of Life?
Summary: RNA/DNA storage invented late in life
Keywords: RNA DNA evolution origin life
Date: 18 Aug 92 19:31:36 GMT
Organization: Duke University Physics Dept.; Durham, N.C.

Quite a few biochemists will argue that RNA and DNA are
storage devices which evolved only after living cells were well
established.  In particular, the intermediate (ancestral)
metabolism was required before RNA and DNA could be used,
since nitrogen-containing compounds are not cheap and easy
to find.

So sayeth Harold Morowitz (book due out soon), and there is
another new book out on the topic of the origins of life
which also refutes the RNA-first theory (sorry, no citation).

	Una
-- 

Una Smith   una@phy.duke.edu    School of the Environment
                                Duke University
                                Durham, NC  27706
From: christa@rocky8.rockefeller.edu (Alan Christensen)
Newsgroups: bionet.molbio.evolution
Subject: Re: exon shuffling question
Date: 21 Aug 92 19:52:01 GMT
Reply-To: christa@rocky8 (Alan Christensen)
Organization: Rockefeller Univ.,N.Y.C 10021

Better still, does anyone know of an example of convergent evolution
that seems to have happened by exon shuffling?  That is, two proteins
of identical or very similar function that do not resemble each other
at all, yet do resemble pieces of other proteins in the databases.
If exon shuffling is really true, then shouldn't this happen?
From: tmhoff@oogoody.Corp.Sun.COM (Todd Hoff)
Newsgroups: bionet.molbio.evolution
Subject: Re: Making Evolution More Efficient
Date: 19 Aug 92 15:51:30 GMT
Reply-To: tmhoff@oogoody.Corp.Sun.COM
Organization: Sun Microsystems, Inc.
NNTP-Posting-Host: oogoody.corp.sun.com

>Hard to speculate until you define what you mean by efficient-
>Fewer cycles to an optimized subroutine?

Take your pick. But what I mean by efficient is to pack more information,
perhaps new characteristics, in the same space, react quicker to external
changes, be more daring in how genes are transfered and selected, and
probably lots more I don't know about. I realize the generally conservative 
approach in our bodies now is probably better, but what would happen if some the 
rules changed?
From: dbd@THEORY.BCHS.UH.EDU (Dan Davison)
Newsgroups: bionet.molbio.evolution
Subject: Postdoctoral Fellowships in molecular evolution
Date: 21 Aug 92 21:49:11 GMT
Distribution: bionet


[Posted by D. Davison; send requests for information to the address
below; the late posting is my fault]

The Alfred P. Sloan Foundation
Postdoctoral Fellowships in Molecular Studies of Evolution
FINAL ROUND

DEADLINE: SEPTEMBER 4, 1992

Awards will support up to two years of research work at a department or
laboratory in the US or Canada selected by the applicant.  The principal
selection criterion will be the potential of the applicant and the proposed postdoctoral research training plan for further molecular-level under-
standing of evolution. The suitability of the institution selected and 
the support for the postdoctoral research indicated by the senior 
scientist (department chair or laboratory director) will be taken 
into account as part of the selection process. Only one proposal 
per applicant can be considered.

There are no formal application forms needed for this program.
Further details and application procedures are available from:

Dr. Michael S. Teitelbaum
Postdoctoral Fellowships
Alfred P. Sloan Foundation
630 Fifth Avenue, Suite 2550
New York, NY 10111
212-649-1649



Up to 10 fellows will be selected.  Stipend is $25,000 to the Fellow,
inclusive of benefits.  $10,000 per year to the sponsoring lab or department,
with the understanding that reasonable research expenses of the Fellow
will be supported from these funds at the discretion of the senior
scientist. Institution may request up to 15% of direct costs for overhead.

ELIGIBILITY:
Fellow: PhD earned within the past 5 years or expected by September 1993
in molecular biology, evolutionary biology, or closely related discipline.
Initiation of postdoc requires completion of PhD).
Newsgroups: bionet.molbio.evolution
From: rees@ocs.mq.edu.au (Rees Griffiths)
Subject: email address for Dr Bernie Crespi ar Cornell?
Originator: rees@laurel.ocs.mq.edu.au
Organization: Macquarie University, Australia.
Date: Thu, 20 Aug 1992 03:06:17 GMT


   email utilities such as netfind found the address crespi@gsm.cornell.edu
but that bounced.
   Does anybody know Dr Crespi's current email address?

Many thanks in advance (and sorry about the waste of bandwidth)
Rees
Newsgroups: bionet.molbio.evolution
From: tmcfarla@fraser.sfu.ca (Tara McFarlane)
Subject: Re: Making Evolution More Efficient
Organization: Simon Fraser University, Burnaby, B.C., Canada
Date: Fri, 21 Aug 1992 11:11:37 GMT

well it would appear that people want to limit their imaginations. now, if you
were an evolutionary epistemologist you would view biological evolution as just
one area of knowledge acquistion. all knowledge systems can be viewed in the 
general darwinian scheme of variation, selection, retension, how valid an 
analysis this is is another matter. anyway, human culture can also be viewed
evolutionarily. i don't think there is much doubt that human culture evolves
much faster than human biology--ok, so no one said that speed of adaptation
is a measure of efficiency. perhaps if you look at the differences between
cultural versus biological evolution with respect to how variation is produced,
how it is selected, and how it is retained, you might come up with the 
answer you are looking for.
From: gwilliam@crc.ac.uk (Gary Williams x3294)
Newsgroups: bionet.molbio.evolution,sci.bio
Subject: Genome size and no. of genes
Keywords: e. coli yeast nematode drosophila mouse human genome size gene number
Date: 17 Aug 92 09:13:46 GMT
Followup-To: poster
Organization: MRC Human Genome Resource Centre

I'm trying to get a more accurate value for the genome size and total
number of genes for various species in the genome mapping projects.

Could anyone provide more accurate figures for any parts of this table?

Species        Genome size       No. of genes

E.coli            5 Mbp              ?
Yeast            15 Mbp              ?
Nematode        100 Mbp              ?
Drosophila      300 Mbp            15,000
Mouse          3000 Mbp        50,000 - 100,000
Human          3000 Mbp        50,000 - 100,000


Many thanks,
-- 
GARY WILLIAMS,  Computing Services Section,  Janet:       G.Williams@UK.AC.CRC
MRC-CRC & Human Genome Mapping Centre,       Internet:    G.Williams@CRC.AC.UK
Watford Rd, HARROW, Middx, HA1 3UJ, UK
Tel 081-869 3294   Fax 081-423 1275
From: colby@bu-bio.bu.edu (Chris Colby)
Newsgroups: bionet.molbio.evolution
Subject: exon shuffling question
Date: 13 Aug 92 02:54:51 GMT
Organization: animal -- coelomate -- deuterostome

According to the theory of exon shuffling, some genes are assembled
over evolutionary time by bringing together "pieces" (exons) of
other genes. Has any one picked an exon that seems common to
many proteins(*), put the sequences into PAUP or PHYLIP and 
constructed a phylogeny? It seems to me you could at least
postulate a sequence of how some exons diverged (and how some genes
evolved?)

A nice cross-check would be to construct the same phylogeny from the 
same exons in another species (assuming the exons diverged prior to 
the species). (You could check for gene conversion this way.)

Selective constraints and gene conversion would probably be
confounding factors along with the fact that the genes may have
diverged long ago (depending upon what exons you pick).

(*) within a single species

So, has anyone done this? Is it even worth doing?

Chris Colby 	---	 email: colby@bu-bio.bu.edu	---
"'My boy,' he said, 'you are descended from a long line of determined,
resourceful, microscopic tadpoles--champions every one.'"
 	--Kurt Vonnegut from "Galapagos"
From: tmhoff@oogoody.Corp.Sun.COM (Todd Hoff)
Newsgroups: bionet.molbio.evolution
Subject: Making Evolution More Efficient
Date: 18 Aug 1992 23:01:30 GMT
Organization: Sun Microsystems, Inc.
Distribution: world
Reply-To: tmhoff@oogoody.Corp.Sun.COM
NNTP-Posting-Host: oogoody.corp.sun.com


How could the mechanisms of evolution be made more efficient?
I've fooled around some with genetic algorithms and it's interesting
to try different strategies. Currently nature uses crossover and
mutation to mix up the genetic soup. What would happen to humans if
we could change how genetic information is passed? There's also
some genetic information that is ignored during mitosis, what would
happen if we could replace the ignored information with good information?
What if we could change the fitness evaluation function as well?

As you've probably guessed I really don't know what I'm talking about,
but I'm trying. Does anyone have any speculations?
From: tomh@BAMBI.CCS.FAU.EDU (Tom Holroyd)
Newsgroups: bionet.molbio.evolution
Subject: Re: Was There Once an RNA World of Life?
Date: 18 Aug 92 20:18:27 GMT
Distribution: bionet

Mmm.  Genes and enzymes evolved later to modulate and regulate
processes that were already going on.  RNA might have been the
first information storage molecule.

But what were those processes like?  Autocatalytic sets?

Tom Holroyd
Center for Complex Systems and Brain Sciences
Florida Atlantic University, Boca Raton, FL 33431 USA
tomh@bambi.ccs.fau.edu
From: mkkuhner@phylo.genetics.washington.edu (Mary K. Kuhner)
Newsgroups: bionet.molbio.evolution
Subject: Re: exon shuffling question
Date: 13 Aug 92 06:17:47 GMT
Organization: University of Washington, Seattle

Phylogenies have been constructed for exons of the MHC genes, but
the problem is that an exon doesn't really contain enough base pairs
to make good phylogenies.  The results approach noise level as soon
as there is much divergence.

M. Nei has at least one paper on this.

Mary Kuhner mkkuhner@genetics.washington.edu
From: nexus@matt.ksu.ksu.edu (Arseny Nikolaev Markov)
Newsgroups: bionet.molbio.evolution
Subject: Re: Was There Once an RNA World of Life?
Date: 18 Aug 1992 15:35:15 -0500
Organization: Kansas State University
NNTP-Posting-Host: matt.ksu.ksu.edu
Keywords: RNA DNA evolution origin life

una@phy.duke.edu (Una Smith) writes:

>Quite a few biochemists will argue that RNA and DNA are
>storage devices which evolved only after living cells were well
>established.  In particular, the intermediate (ancestral)
>metabolism was required before RNA and DNA could be used,
>since nitrogen-containing compounds are not cheap and easy
>to find.

>So sayeth Harold Morowitz (book due out soon), and there is
>another new book out on the topic of the origins of life
>which also refutes the RNA-first theory (sorry, no citation).

>	Una
>-- 

>Una Smith   una@phy.duke.edu    School of the Environment
>                                Duke University
>                                Durham, NC  27706


Did God made the enzymes for the intermediate metabolism? How got those
"living cells" well established without a heritage information?
 ^^^^^^
And were they living without the ability to pass information to a
"progeny"?

inquiring minds want to know,

the NEXUS>
Newsgroups: bionet.molbio.hiv
From: bz965@cleveland.Freenet.Edu (Richard Hatch)
Subject: Purpose of this group?
Organization: Case Western Reserve University, Cleveland, Ohio, (USA)
Date: Sat, 15 Aug 92 03:08:43 GMT


What is the purpose of this group? molbio of HIV is a pretty 
weighty subject, but I don't see a lot of postings. Is there 
perhaps another group somewhere, where things like phylogenetics
are being discussed? Does anyone know?
From: CLARK@SALK-SC2.SDSC.EDU
Newsgroups: bionet.molbio.hiv
Subject: Re: Some HIV PCR primers
Date: 17 Aug 92 17:23:01 GMT
Distribution: bionet

/Some time ago I suggested the start of a project that would compile PCR primers
/useful in HIV-reserarch. At that time I had in mind a rather sophisticated
/database that would present various data about the primers, such as the
/references where they had been used, the optimal conditions they had been used
/at, their sensitivity and specificity for different strains, the combinations
/they could be used in etc, etc.
/
/However as things ususally turned out, there was some interest, but I
/underestimated the work to be done for the collecting of these data.
/ [remainder deleted]

Not too long ago there was a posting on one of the Biosci groups about a
group in Italy that has set up a Molecular Probe Database which sounds like 
it would be a superset of what you want. A search by WAIS of the biosci
messages archived at GenBank was unfortunately unable to turn up the
original message :-( , but I have an email address of someone who could
provide more information. It is ipus@istge.ist.unige.it (Interlab Project 
Users Service).


Steve Clark

clark@salk-sc2.sdsc.edu  (Internet)
clark@salk               (Bitnet)
From: kristoff@GENBANK.BIO.NET (Dave Kristofferson)
Newsgroups: bionet.molbio.genbank
Subject: Re: Getting Sequences Published by an Author
Date: 12 Aug 92 19:46:07 GMT
Distribution: bionet


I would highly recommend NCBI's retrieval server over the use of
gopher, etc., for the purpose requested.

Dave Kristofferson
From: michael@TEMIN.LANL.GOV (Michael J. Cinkosky)
Newsgroups: bionet.molbio.genbank
Subject: Re: GenBank error?! (brain damage?)
Date: 12 Aug 92 22:31:26 GMT
Distribution: bionet


> Well, I wrote a program to fish out coding exons from GenBank by looking
> at the 'CDS join...' entry in the feature table and came out with this 
> interesting entry which appears to contain a 3 bp internal exon (and an intron of 
> length zero!)...  
>
>     CDS             join(3455..3511,3512..3515,5060..5092,6258..6333,
>                     7025..7166)
>
> What is the point of 'joining' the sig_pep separately?  I would have liked to 
> think that the purpose of 'join' was to link segments which are non-contiguous...
> Are signal peptides always treated this way or is this just an odd ball case?
> ...
> ---------------------------------------------------------------------------
> TTGATTGCTAAACACTGGGCGGCGAATCAGGGTTGGGATCTGAACAAAGACGGTCAGATTCAGTTCGTACTGCTG
> Eric E. Snyder                            
> Department of MCD Biology              ...making feet for childrens' shoes.
> University of Colorado, Boulder   
> Boulder, Colorado 80309-0347
> LeuIleAlaLysHisTrpAlaAlaAsnGlnGlyTrpAspLeuAsnLysAspGlyGlnIleGlnPheValLeuLeu
> ---------------------------------------------------------------------------



Eric,

Like any complex language, there are many ways of making any
one statement in the GenBank/EMBL/DDBJ feature table format.
This assumption that you made that a join() would only be
used to join non-contiguous segments together (most notably,
exons), is not a requirement of either the syntax of the language
or of its preferred usage.  (Essentially, there is no statement
about this in our standard usage guides here.)

Further, while one might certainly propose a guideline such as
this for future annotation, I am not sure that it would be a good
idea.  It is often the case that annotators (including those who
submit their data to us directly through Authorin) find it very
convenient to create larger features (such as the CDS in the entry
you mentioned) out of smaller pieces (such as the sig_peptide
and mat_peptide).  This saves considerable effort, both in
original annotation as well as in maintenance (e.g., if we
later detect that the sig_peptide is incorrect, whatever changes
we make will be automatically reflected in the CDS). 

So, while it does mean that one needs to check to make sure that
the next piece of a join() is non-contiguous with the current piece,
I would argue that this is a small price to pay for the ability to
build larger features from smaller ones.

Michael Cinkosky
GenBank / Los Alamos