HDTenEightyP/NetNews huggingface dataset:
From: lpcasson@phoenix.Princeton.EDU (Lawrence P. Casson)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Nucleotide analogs and PCR mutagenesis
Date: 26 Jan 93 20:02:22 GMT
Organization: Princeton University
Originator: news@nimaster

I am investigating various mutagenesis methods with the goal of
randomly introducing multiple changes into a cloned gene.
In particular, I am interested in the PCR (am I allowed to say PCR?)
based technique described by Leung, D.W., et al. (Technique 1:11-15)
and Cadwell, R.W., and Joyce, G.F. (PCR Methods and Applications 2:28-33).

I am interested in the use of nucleotide analogs in combination with
this technique.  For example, DNA sequencing is often done with dITP
or 7-deaza-dGTP because the weaker base pairing interactions eliminate
certain gel artifacts.  Since the PCR mutagenesis technique reduces
the fidelity of the Taq polymerase,  these nucleotides might be
incorporated at a high rate.  Perhaps this could be extended to other
nucleotide analogs.  The major benefit would be a larger number of
mutations in fewer PCR cycles.

The ideal "universal" nucleotide analog will pair with any other
nucleotide and be randomly incorporated and not cause a large number
of frame shift mutations.  However, I am willing to settle for
second best.

Does anyone know of any work regarding nucleotide analogs and base
pairing?  Has anyone used analogs to investigate kinetics of various
DNA or RNA polymerases?


Larry Casson
lpcasson@phoenix.princeton.edu
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Looking for non-coli vector
Date: 28 Jan 1993 05:21:53 GMT
Organization: University of Houston
Distribution: bionet
Reply-To: bchs1b@Elroy.UH.EDU
NNTP-Posting-Host: elroy.uh.edu

In article <9301272018.AA19221@umailsrv0.UMD.EDU>, William_D_WARREN@UMAIL.UMD.EDU (ww40) writes:
>G'day,
>
>We are looking for a double stranded, closed circular DNA vector
>that will not replicate in E coli.
>
>What is needed is a system whereby we can do invitro manipulation and
>easily reintroduce the DNA back into it's host which will then grow it
>up with high fidelity so we can isolate large quantities (around 1-200ug)
>of monomeric molecules. We are currently considering B subtilis plasmids
>however their propensity for concatamerization, instability and poor
>transformation are far from ideal for our purposes. What is important,
>as mentioned above, is that they not replicate in E coli and are double
>stranded closed circles.
>
>Does anyone out there have any suggestions ???
>
>BILL WARREN
>Center Ag Biotech
>University of Maryland
>
>

It isn't exactly clear what you want to do. But as an alternative, how
about a plasmid which selectively replicates only in certain E. coli
strains or under certain conditions. There are some vectors requiring 
specific replication proteins which need to be provided in trans, usually
from an lambda lysogen. So the plasmid only replicates in these hosts
and not in standard E.coli. I think these are PIR plasmids or something
similar. I can get some references when in my office if you want.
Alternately, there is a vector whose replication is tightly controlled
by lac repressor such that it only replicates in the presence of IPTG.
Would something like this serve your purpose better than using
heterologous strains???

----------------------------------------------------------------------
 Michael Benedik				INTERNET: Benedik@uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik@uhou
-----------------------------------------------------------------------
From: rideout@methylc.hsc.usc.edu (William Rideout)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Shuttle vector- Hirt lysate yields
Date: 23 Jan 1993 01:58:08 -0800
Organization: University of Southern California, Los Angeles, CA
NNTP-Posting-Host: methylc.hsc.usc.edu
Keywords: Hirt lysates, episomal DNA purification

Hello,

I could use some suggestion for improving my Hirt lysate yield of 
episomal DNA from EBV+ lymphocytes.  I am using a vector that 
replicates as an episome and need to get as many bacterial colonies
as possible after electroporation of the Hirt lysate DNA into the 
appropriate bacterial strain.

EtBr stained agarose gels indicate a fair amount of contaminating 
sheared or degraded genomic DNA.  I fear that this material is reducing
my electroporation efficiency.  Blotting and probing of these gels indicates
that the majority of episomal DNA is intact as covalently-closed circles. 

Any advice as to improve my yield (10,000 colonies max right now from an
estimated amount of 1ng of episomal shuttle vector).

Any advice would be most welcome. I am currently following the gerneral
procedure of Hirt himself.

Frustratedly yours,

britt
rideout@zygote.hsc.usc.edu
From: jdboer@SOL.UVIC.CA (Johan deBoer)
Newsgroups: bionet.molbio.methds-reagnts
Subject: TCR of B-cells?
Date: 27 Jan 93 07:20:13 GMT
Distribution: bionet

Hello M&R's,

This is a question directed to the more immunologically oriented. We are
using human T-cells and B-cells for the study of mutations. In order to
detect clonal relationships between cells I have devised a method for 
T-cells that PCRs a fragment comprising the variable and joining region of 
the rearranged gamma receptor gene. A restriction digest then will tell
us possible clonal relationships.

For B-cells we would probably have to rely on the alpha and beta genes. 
There are however many more segments from which the cell can pick during
the rearrangement and this makes PCR (whic requires some conserved sequence)
a whole lot more difficult. Has anyone ever tried something like this for
B-cells or does someone have any ideas regarding this?

Any comments would be appreciated.

Johan de Boer
jdboer@sol.uvic.ca
University of Victoria, Canada
From: wsun@jeeves.ucsd.edu (Fiberman)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: insert-positive blue cols
Date: 23 Jan 93 23:15:35 GMT
Organization: University of California, San Diego

In article <1993Jan21.152404.26275@magnus.acs.ohio-state.edu> cbreiten@magnus.acs.ohio-state.edu (Caroline A Breitenberger) writes:

[stuff deleted]
>
>In another case, a graduate student who is using the Clontech TA cloning
>kit (:-)) noted that clones containing his desired PCR-derived insert
>(appr. 250 bp) exhibit a pale blue phenotype.  In this case, I think
>that the insert provides a continuous open reading frame which results
>in expression of the alpha-complementation peptide with an N-terminal
>extension.  The pale blue color suggests that this peptide is less
>efficient than that of the parental vector.  (The opposite orientation
>of the insert presumably gives white colonies - I forgot to ask him.)
>
>Caroline Breitenberger, Department of Biochemistry
>Ohio State University, 484 W. 12th Ave., Columbus, OH 43210, USA
>614-292-9473.......caroline+@osu.edu


I have had the same experience.  I tried calling Stratagene,
asking them where does the alpha complemetation peptide start in
the bluescript vector and how long is the peptide, but the
technician did not know anything.  If the peptide is short
enough, cloning an insert into a site that's at the 3' end of
the polylinker will not give you any white colonies.  Any one
out there knows how long is the alpha comlementation peptide?

-fm
From: kh@litsun17.epfl.ch (Mark S.)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: western blotting advice -ECL
Date: 28 Jan 93 17:59:34 GMT
Reply-To: kh@litsun.epfl.ch
Distribution: bionet
Organization: Ecole Polytechnique Federale de Lausanne
In-reply-to: fgarbrec@POST.ITS.MCW.EDU's message of 19 Jan 93 23:34:28 GMT


    > I have been using the ECL  kit  from Amersham to do some western
    > blotting, but it seems almost too sensitive.  I get all kinds of
    > background   (not    the   diffuse   kind    from   insufficient
    > blocking/washing) but jillions of specific  bands that shouldn't
    > really be  there.  Has anyone experiance with  this application,
    > and/or  suggestions  as how to  diminish the background problem?

I usually do blocking (milkpowder) overnight and incubate first anti-
body at least 4 h. This does not solve the problem completely but the
results are much more clear.

please reply to dj02@dkauni2.bitnet.
--

-------------------------------------------------------------------------
Mark Soekarjo				Living in a postcard

postcards to 	la Maison Rose		e-mail to	kh@litsun.epfl.ch
		Ch. des Clos 53		fax		+41'21'693'41'11
		CH-1024 Ecublens	voice		+41'21'693'34'83
		****europe*****         canned voice    +41'21'693'34'83
Newsgroups: bionet.molbio.evolution
From: mwooten@ducvax.auburn.edu
Subject: Anti-G bias question
Organization: Auburn University, AL
Date: Fri, 22 Jan 1993 14:29:09 GMT

I am new to this newsgroup so I apologize if this issue has been worked through
before.  

Issue:  In working with mitochondrial Cytochrome B sequences from livebearing
        fish, I have noted the same strong anti-G bias that has been reported
        for numerous mammals and other taxa.  My fish show somewhat less of a
        bias but the values are still significant and very consistent with
        patterns in other coding regions (both nuclear and mitochondrial).
        The most obvious location of this bias is in the third position where
        G is found less than 8% of the time.

Question: What molecular mechanism(s) could account for this pattern?

I have tried to work through a number of possible explanations based on
G-C binding and hairpins during transcription but given these ideas, it
is still unclear to me how any bias arises in the first place.

Anyone interested in a discussion on this issue?

Mike

***********************************************************************
*
*   Michael C. Wooten
*
*   Dept. of Zoology
*   Auburn University
*   Auburn, AL  36849  USA
*
*   BITNET: mwooten@auducvax.bitnet
*   INTERNET: mwooten@ducvax.auburn.edu
*
***********************************************************************
From: kristoff@NET.BIO.NET (David Kristofferson)
Newsgroups: bionet.molbio.evolution
Subject: BIOSCI/bionet Frequently Asked Questions
Date: 21 Jan 93 10:00:02 GMT
Distribution: bionet


New users of BIOSCI/bionet may want to read the "Frequently Asked
Questions" or "FAQ" sheet for BIOSCI.  The FAQ provides details on how
to participate in these forums and is available for anonymous FTP from
net.bio.net [134.172.2.69] in pub/BIOSCI/biosci.FAQ.  It may also be
requested by sending e-mail to biosci@net.bio.net (use plain English
for your request).  The FAQ is also posted on the first of each month
to the newsgroup BIONEWS/bionet.announce immediately following the
posting of the BIOSCI information sheet.

				Sincerely,

				Dave Kristofferson
				BIOSCI/bionet Manager

				kristoff@net.bio.net
From: HABERMANN@AIMP.UNA.AC.AT
Newsgroups: bionet.molbio.embldatabank
Subject: promotor and database-search?
Date: 27 Jan 93 16:13:00 GMT
Distribution: bionet
Original-To: embl-db@uk.ac.daresbury
X-Vms-To: BITNET"embl-db@daresbury.ac.uk"

To the EMBL-DATABANK,  bionet.molbio.embldatabank

Suppose you had promotor sequences of several - functionally related - genes with a conserved sequence 
motif in common, 
1) how could you find out, if there is already a binding protein known for this specific motif?
2) how would you look for promotors of other genes containing the same conserved sequence element?
I know that there exists an Eukaryotic Promotor Database - somewhere out there - which might even be 
useful. I also heard that this EPD is available on the EMBL-server. Is there any possibility to access this 
database? If yes, how?
If you have any ideas how to solve this problem - apart from performing a fasta with this sequence and 
looking through the tons of output-datas by hand, we had this idea already and are not thrilled with the 
thougt, since a fasta-search of about 20 nucleotides against the GenEMBL database overwhelms our 
imagination - please let me know!
Thanks in advance
Bianca

-----------------------------------------------------------------------------------------------------------------

e-mail address (bitnet) :
habermann@aimp.una.ac.at
institute of molecular pathology,  Dr.Bohrgasse7, A1030 Wien

_________________________________________________________________________________
From: toms@fcs260c2.ncifcrf.gov (Tom Schneider)
Newsgroups: bionet.molbio.embldatabank
Subject: Re: promotor and database-search?
Date: 28 Jan 93 01:15:57 GMT
Distribution: bionet
Organization: Frederick Cancer Research and Development Center

In article <1993Jan27.182937.12863@gserv1.dl.ac.uk>
Bianca (HABERMANN@AIMP.UNA.AC.AT) writes:

| Suppose you had promotor sequences of several - functionally related - genes
| with a conserved sequence motif in common,

Two sequence recognizers can have identical consensus sequences, and yet
recognize different patterns.  Therefore you should not use consensus
sequences.  Instead, make sequence logos of the sequences you have at hand and
compare them to the sequence logos of other sites.  The results will be highly
sensitive and mathematically much more sensible.

See:

@article{Stephens.Schneider.Splice,
author = "R. M. Stephens
  and T. D. Schneider",
title = "Features of spliceosome evolution and function
inferred from an analysis of the information at human splice sites",
journal = "J. Mol. Biol.",
volume = "228",
pages = "1124-1136",
year = "1992"}

Regards,

  Tom Schneider
  National Cancer Institute
  Laboratory of Mathematical Biology
  Frederick, Maryland  21702-1201
  toms@ncifcrf.gov
From: demirov@genes.icgeb.trieste.it (Dimter Demirov)
Newsgroups: bionet.molbio.hiv
Subject: AIDS gene therapy
Summary: Looking for Foundations for AIDS research
Keywords: AIDS
Date: 28 Jan 93 10:36:29 GMT
Organization: ICGEB

I am a postdoctoral fellow very much interested in developing a
project for AIDS gene therapy. As I have a project ready I would
like to know names and addresses of foundations for AIDS research
so as to apply for a grant.

I will be very grateful for any information.

Dimiter Demirov
-------------------------------------------------------------------------
Contact: Dimiter Demirov
  email: demirov@icgeb.trieste.it
Address: ICGEB - International Centre for Genetic Eng. and Biotechnology
         Padriciano 99, I-34012 Trieste Italy
From: ostell@object.nlm.nih.gov (Jim Ostell)
Newsgroups: bionet.molbio.genbank
Subject: Re: A continuing mis-use of misc_feature?
Date: 25 Jan 93 18:15:34 GMT
Organization: National Library of Medicine
X-Newsreader: Tin 1.1 PL4

The problem Keith Robison has noticed is a consequence of the differing
data models specified by the ASN.1 spec NCBI uses to unify all the
data sources we integrate, and the peculiarities of the GenBank flatfile
format. In the ASN.1, there is a Gene feature which defines a region
of nucleic acid. That gene region may have other features over the same
interval, such as coding regions or tRNAs. In the GenBank flatfile, the
concept of a gene only exists as a qualifier /gene, on some other feature.
Not all features are allowed to have the gene qualifier, according to
the GenBank/EMBL/DDBJ feature table document.

the sequence database can produce.  It is very important that you
realize the flatfile is not the database... when you see things in the
flatfile that aren't so great, we want to know about them, but it does
not follow that the database itself is "junked up".

Now, since gene only exists as a qualifier on other features in the flatfile,
what do we do when, in the underlying database we know where a gene is and
what its name is, but there doesn't happen to be a CDS to make a GenBank
style qualifier on? Rather than throw the information away for those of
you still looking at the flatfile, we opted to create a misc-feature and
put a gene qualifier on it.  Not beautiful, but at least you flatfile fans
can still see the data. The real solution would be to start looking at the
ASN.1 of course, but we do want to be as backward compatible as possible.

That explains most of the misc_feature you have seen this way.  You are
also seeing other effects of the conversion.  For example, a CDS for a
pseudogene which is full of stops is not a CdRegion (which is a protein
coding region... i.e. codes for a real protein). So those are not converted
to CdRegion features in the ASN.1.  They reappear in the flatfile, but
are not treated internally the same as a CdRegion.  So the flatfile
generating code mistakenly did not put the gene qualifier back on it
when producing the flatfile. There were a couple other cases of this
sort of mismapping in the flatfile generator.  Happily those problems
were easily fixed (actually we noticed the same problem, and these fixes
were made a month ago).  So you should not see these cases in the next
flatfile release.  Just let us know (politely) if you do see some.

So, unless we hear a strong vote against it, we will continue to produce
misc-features in the GenBank flatfile when we have no legal other feature
to put a gene qualifier on, but when we know the gene and can't express that
information directly given the flatfile format restrictions.

We want to hear about any problems with the flatfile report, but remember
that is only a report.  If you want to look at the real underlying data,
look at the ASN.1. There you will see not only the DNA sequences and
features you see in the flatfile, but also proteins, and other data which
cannot be well represented in the flatfile.


  Jim Ostell
  NCBI
From: ostell@object.nlm.nih.gov (Jim Ostell)
Newsgroups: bionet.molbio.genbank
Subject: Re: A continuing mis-use of misc_feature?
Date: 25 Jan 93 18:27:19 GMT
Organization: National Library of Medicine
X-Newsreader: Tin 1.1 PL4

The first line of the second paragraph in my response was dropped. It
is

You must realize the GenBank flatfile is just one of many reports the ...


  Jim Ostell
  NCBI
From: GenBank-Updates@net.bio.net
Newsgroups: bionet.molbio.genbank.updates
Subject: Rice DNA, partial sequence of RFLP marker.
Date: 22 Jan 93 19:48:37 GMT
Distribution: bionet
Checksum: 54991 2

LOCUS       RIC037GN22    296 bp ds-DNA             PLN       22-JAN-1993
DEFINITION  Rice DNA, partial sequence of RFLP marker.
ACCESSION   D13523
KEYWORDS    RFLP marker; STS sequence;
            restriction fragment length polymorphism; sequence tagged site.
SEGMENT     2 of 3
SOURCE      Oryza sativa (strain Nipponbare, sub_species Japonica) DNA, clone
            GN37.
  ORGANISM  Oryza sativa
            Eukaryota; Plantae; Embryobionta; Magnoliophyta; Liliopsida;
            Commelinidae; Cyperales; Poaceae.
REFERENCE   1  (bases 1 to 296)
  AUTHORS   Minobe,Y.
  TITLE     Nucleotide sequence of sequence tagged site from rice RFLP marker
            probe
  JOURNAL   Unpublished (1992)
  STANDARD  full automatic
COMMENT     Submitted (29-Oct-1992) to DDBJ by: Yuzo Minobe Rice Genome
            Research Program National Institute of Agrobiological Resources
            2-1-2 Kannondai, Tsukuba Ibaraki 305 Japan Phone:  0298-38-7441
            Fax:    0298-38-7468 email:  MINOBE@rtcs0.riken.go.jp.
From: GenBank-Updates@net.bio.net
Newsgroups: bionet.molbio.genbank.updates
Subject: Ig V lambda IV=immunoglobulin V lambda [human, Genomic, 632 nt]
Date: 27 Jan 93 06:00:02 GMT
Distribution: bionet
Checksum: 29200 3

LOCUS       S119586       632 bp    DNA             PRI       21-JAN-1993
DEFINITION  Ig V lambda IV=immunoglobulin V lambda [human, Genomic, 632 nt]
ACCESSION   S49668 M94113
KEYWORDS    .
SOURCE      human
  ORGANISM  Unclassified.
            Unclassified.
REFERENCE   1  (bases 1 to 632)
  AUTHORS   Daley,M.D., Peng,H.Q., Misener,V., Liu,X.Y., Chen,P.P. and
            Siminovitch,K.A.
  TITLE     Molecular analysis of human immunoglobulin V lambda germline genes:
            subgroups V lambda III and V lambda IV.
  JOURNAL   Mol. Immunol. 29, 1515-1518 (1992)
  STANDARD  full automatic
COMMENT     This entry [NCBI gibbsq 119586] was created by the journal scanning
            component of NCBI/GenBank at the National Library of Medicine.
            This sequence comes from Fig. 1.  The authors begin their numbering
            at -302.  Zero is not present in the numbering.
FEATURES             Location/Qualifiers
     CDS             300..587
                     /partial
                     /note="For the protein sequence (NCBI gibbsq 119587):
                     Method: conceptual translation supplied by author.  This
                     sequence comes from Fig. 2."
                     /gene="Ig V<lambda>IV"
                     /product="immunoglobulin V lambda"
                     /codon_start=1
                     /translation="SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPV
                     LVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNH"
BASE COUNT      141 a    179 c    162 g    150 t
ORIGIN      
        1 atttgcatga agagacttcc cttcctatga taagagaggc ctggaggttc ctccttagct
       61 gtgggctcag aagcagagtt ctggggtgtc tccaccatgg cctggacccc tctctggctc
      121 actctcctca ctctttgcat aggtgctgcc tcccagggct caaccccata ttatcatgct
      181 agctgtgcca acctggccct gagcttcggc tcaacacagg gagtagtgta gggtgtggga
      241 ctctaggcgt gaaaccctta tcctcacctc ttctgtcctc ttttgcaggt tctgtggttt
      301 cttctgagct gactcaggac cctgctgtgt ctgtggcctt gggacagaca gtcaggatca
      361 catgccaagg agacagcctc agaagctatt atgcaagctg gtaccagcag aagccaggac
      421 aggcccctgt acttgtcatc tatggtaaaa acaaccggcc ctcagggatc ccagaccgat
      481 tctctggctc cagctcagga aacacagctt ccttgaccat cactggggct caggcggaag
      541 atgaggctga ctattactgt aactcccggg acagcagtgg taaccatctc acagtgacac
      601 agacagatgg ggaagtgaga cagaaacccc tt
//
From: GenBank-Updates@net.bio.net
Newsgroups: bionet.molbio.genbank.updates
Subject: trpE {promoter} [plasmid pATH10.1, Plasmid Recombinant, 400 nt]
Date: 28 Jan 93 05:48:32 GMT
Distribution: bionet
Checksum: 42846 2

LOCUS       S120197       400 bp    DNA             BCT       27-JAN-1993
DEFINITION  trpE {promoter} [plasmid pATH10.1, Plasmid Recombinant, 400 nt]
ACCESSION   S49970
KEYWORDS    .
SOURCE      plasmid pATH10.1
  ORGANISM  Unclassified.
            Unclassified.
REFERENCE   1  (bases 1 to 400)
  AUTHORS   Mercy,M.R., Troncoso,J.C. and Monteiro,M.J.
  TITLE     A new series of trpE vectors that enable high expression of
            nonfusion proteins in bacteria.
  JOURNAL   Protein Expr. Purif. 3, 57-64 (1992)
  STANDARD  full automatic
COMMENT     This entry [NCBI gibbsq 120197] was created by the journal scanning
            component of NCBI/GenBank at the National Library of Medicine.
            This sequence comes from Fig. 2.
FEATURES             Location/Qualifiers
     CDS             398..400
                     /partial
                     /note="Only start codon shown"
                     /gene="trpE"
BASE COUNT      112 a     90 c     99 g     99 t
ORIGIN      
        1 gccagggtgc cgacgcgcat ctcgactgca cggtgcacca atgcttctgg cgtcaggcag
       61 ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc
      121 aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg atctggcaaa
      181 atatctgaaa tgagctgttg acaattaatc atcgaactag ttaactagta cgcaagttca
      241 cgtaaaaagg gtatcgacaa tgaaagcaat tttcgtactg aaaggttggt ggcgcacttc
      301 ctgaaacggg cagtgtattc accatgcgta aagcaatcag atacccagcc cgacgctaat
      361 gagcgggctt ttttttgaac aaaattagag aataacaatg
//