\documentstyle{dragnlab98}
\me{Robin.Treichel@oberlin.edu}

\newcommand{\kappa}{${}_\image{\icondir kappa.gif}$}
\seticondir{../icons/}
%\solutionon
\lib{igg}{Definitions}
\title{Immunoglobulin Genes}
\author{Robin S. Treichel}
\wallpaint{aaaaff}

\begin{document}
\maketitle

\header{2}{The Reference}

Evidence for somatic rearrangement of immunoglobulin genes coding for
variable and constant regions.  Nobumichi Hozumi and Susumu Tonegawa.
Proc. Natl.  Acad. Sci. USA 73(10): 3625-3632, 1976.
          
\chapter{Background}

Prior to 1976, indirect evidence revealed that the genome contained
only a few copies of kappa light chain constant-region gene sequences
but contained many separately encoded kappa variable-region gene
sequences.  Immunologists realized some mechanism must integrate the
constant region and variable region information in order to generate a
single continuous protein sequence.  Immunoglobulin-encoding mRNA was
shown to contain both V- and C-region sequences; thus researchers
reasoned that the V- and C-region information could be integrated at
either the DNA or the RNA level.
     
In 1976 Tonegawa and Hozumi reported the first direct evidence that
the information for a single immunoglobulin light chain is encoded by
separate V- and C- region gene segments and that these gene segments
recombine at the level of DNA during the course of B cell
differentiation.  Their discovery challenged the biological dogma of
the time because (1) it contradicted the one gene-one polypeptide
principle, and (2) it contradicted the idea that the genome is
constant during ontogeny and cell differentiation.  For this
ground-breaking discovery, Susumu Tonegawa was awarded the 
\zlink{http://www.almaz.com/nobel/}{Nobel Prize} in 1987.

\chapter{Goal and Experimental Approach}

Tonegawa and Hozumi compared DNA from a mouse embryo with DNA from a
plasmacytoma (a plasma cell tumor which produces antibody molecules of
a single type and specificity).  They reasoned that if the V- and
C-region genes had undergone recombination in the tumor cells, the DNA
surrounding those genes should be arranged differently from the DNA of
the embryonic cells and that this difference could be detected using
restriction endonucleases.  If, for example, the C-region gene in
embryonic DNA was bracketed by two restriction sites (the sequences of
DNA cleaved by particular endonuclease) 5000 nucleotides apart,
digestion with that restriction enzyme would generate a fragment 5000
nucleotides long.  However, if prior recombination of V- and C-region
sequences eliminated one of the sites, digestion of the plasmacytoma's
DNA would yield a C-region fragment either longer or shorter than
5,000 nucleotides.

\begin{center}\image{\icondir igrestr1.gif}\end{center}

Using this strategy, Tonegawa and Hozumi were able to show that the
arrangement of -chain genes in embryonic cells differs from the
arrangement in antibody-producing cells.  The basic procedure they
used was as follows:

\begin{enumerate}
\item Messenger mRNA for \kappa chains was purified from the MOPC 321
plasmacytoma.  Part of this mRNA preparation was used to generate a
fragment consisting of the 3'-end half.  The intact mRNA and the 3-end
half mRNA were radioactively labeled with I125.  The labeled mRNA and
the 3-end fragment were prepared so that they could be used as probes
for the light chains gene sequences.

\begin{mcquest}
What gene segments of -light chains will be detected if the entire
mRNA is used as the probe during hybridization ?

\begin{itemize}
\item V gene sequences  
\item D gene sequences  
\item J gene sequences
\item C gene sequences
\end{itemize}
     
\mans{V, J and C}
You are absolutely correct.  
\mans{V and C}
These sequences are present in the entire mRNA, but you are
forgetting a segment that contributes to diversity.  
\mans{V or C}
These sequences are present in the entire mRNA, but you are
forgetting a segment that contributes to diversity.  
\mans{D}
No, D gene sequences contribute to the diversity of heavy chains but not
to that of the light chains of immunoglobulins.
\end{mcquest}


\begin{mcquest}
1b.  What gene segments of  -light chains will be detected if the
3'-end half fragment mRNA is used as the probe?

\begin{itemize}
\item V gene sequences  
\item D gene sequences  
\item J gene sequences
\item C gene sequences
\end{itemize}

\mans{V}
No, the V gene sequence is located at the 5' end of the intact mRNA and
would not be present in the 3' half.
\mans{D}
No, D gene sequences contribute to the diversity of heavy chains but not to
that of the light chains of immunoglobulins.
\mans{J}
The 5' half of the light chain mRNA encodes the amino half of the
light chain.  This portion is specified by the V and J gene segments.
So, the 3' half mRNA would not contain a J gene sequence and so would
not hybridize to J gene segments in the DNA.
\mans{C}
You are correct!  The constant gene sequences would be the only gene
segment detected by this probe.]
\end{mcquest}

\item DNA from MOPC 321 was digested using the restriction enzyme BamH I. 
This enzyme was selected because it cleaved the DNA into a limited
number of fragments of various sizes.

\item DNA from mouse embryos was digested with BamH I.

\begin{exercise}
Which source of DNA is expected to have immunoglobulin genes in the
germline configuration?  Which source is expected to have
immunoglobulin genes in a rearranged configuration?

\begin{exsol}
Embryo DNA is in germline configuration
% give more detail.
\end{exsol}

\end{exercise}

\item Digested DNA preparations were subjected to agarose gel
electrophoresis in order to separate fragments according to size.

\item Fragments in the DNA digest preparations that hybridized with
the radioactively labeled probes were identified.

{\bf Note:  Additional information regarding electrophoretic DNA
separation and hybridization can be found on pages 32-37 of Kuby,
Immunology, 3rd Edition.}
\end{enumerate}

\chapter{Results}

Hybridization with the  3'-half mRNA probe revealed fragments of the
following molecular weights:
\begin{quote}
Embryo:  one fragment, 6.0 x 106 bases

MOPC 321 tumor cells: one fragment, 2.4 x 106
\end{quote}

Hybridization with the entire mRNA probe for  -light chains revealed the
following: 
\begin{quote}
Embryo: two fragments (3.9 x 106, 6.0 x 106)

MOPC321 tumor cells: one fragment (2.4 x 106)
\end{quote}

\begin{gloss*}{Interpretation} 
% ighybri1.gif
\end{gloss*}
\image{\icondir smenaw.gif}

\chapter{Test Your Understanding}

\begin{exercise}
Tonegawa and Hozumi used a very labor intensive approach in order to
determine which DNA digest fragments hybridized with each of the two
probes.  During the time they were performing their experiments the
Southern Blot analysis technique was developed.  In ***Southern Blot
analysis*** the DNA fragments are separated by agarose gel
electrophoresis.  The size-separated DNA is then transferred to a
nitrocellulose filter so that the fragments maintain the same physical
relationship to each other.  The DNA is denatured ("melted" to
separate the two strands of the DNA helix) while still on the
nitrocellulose filter and hybridized to a radiolabeled probe to
identify the fragments containing sequences complementary to the
probe.  In Tonegawa's experiment the restriction enzyme fragments are
identified by their molecular weights.

Use the diagram below to draw the hybridization results Tonegawa and
Hozumi would have observed, if they had been able to use Southern Blot
analysis.  Take into account that two preparations of DNA (embryo and
MOPC321), and two probes were used (entire mRNA and 3-end half).

{\bf Check here if you wish to print this diagram to use as a worksheet.}
\end{exercise}

\begin{exercise}
Indicate on the diagram below your interpretation as to how the entire
mRNA probe would hybridize with each of the DNA fragments detected for
embryo and MOPC tumor cells by Tonegawa and Hozumi (fragment sizes 3.9
X 106; 6.0 X 106; 2.4 X 106).
\end{exercise}

\begin{exercise}
What gene segments are present within the 3.9 kb fragment obtained
from embryo DNA?  What gene segments are present within the 6.0 kb
fragment obtained from embryo DNA?  What gene segments are present
within the 2.4 kb fragment obtained from plasmacytoma DNA?  [Make
certain that you understand why the results obtained with the embryo
DNA using the 3'-end fragment probe are different from those obtained
using the entire mRNA probe, whereas those obtained with MOPC321 DNA
are identical with the two probes.]
\end{exercise}

\chapter{Allelic Exclusion}

DNA from two human myelomas was examined by Southern blot analysis
to investigate the mechanism of allelic exclusion.  The DNA was
cleaved with a restriction enzyme, fragments were separated by agarose
gel electrophoresis, transferred to a nitrocellulose filter, and
hybridized to a radiolabeled probe consisting of sequences
complementary to the J gene segments.  The sizes of these fragments
are indicated in kilobase pairs (kb).

Refer to these results in order to answer the following questions:

\begin{exercise}
Explain the results observed with the sperm DNA.  (What is the
size of the restriction fragment to which the probe hybridizes?  Has a
rearrangement taken place?  Has one or both homologous chromosomes of
the sperm donor undergone a rearrangement of genes?)  

\begin{hint}{Haploid or Diploid?}
Although each sperm is haploid, billions of sperm cells would be used
to isolate DNA.  Thus, both copies of chromosome 2 would be
represented within the DNA preparation and both of the donor's alleles
would be assayed.
\end{hint}
\end{exercise}

\begin{exercise}
Explain the results observed with myeloma 1.  (Has a rearrangement
taken place?  Have one or both chromosomes undergone rearrangement?)
\end{exercise}

\begin{exercise}
Explain the results observed with myeloma 2.  
\end{exercise}

\begin{exercise}
What do these results suggest regarding the phenomenon of "allelic
exclusion"? 
\end{exercise}

\chapter{Drawing Conclusion From Separate Experimental Analyses}

The **Northern blot*** technique is used to examine the transcription
of particular genes and the size of the gene transcripts.  In this
procedure, mRNA molecules that carry poly(A) tails are isolated from
total cellular RNA by adsorption to oligo(dT) cellulose and are
subjected to electrophoresis in agarose under denaturing conditions.
The mRNAs are thus separated by size and can be "blotted" directly
onto nitrocellulose.  Transcripts of interest are identified by
hybridization of radiolabeled probes to the mRNA immobilized on the
nitrocellulose filter.

Northern blots were performed using mRNA from three B cell lineage
tumors B1, B2, B3.  The probe represented the Cæ1 exon of the Cæ
gene.  The characteristics of these tumors are shown in the table
below.  Also shown is the Northern blot performed using mRNA from the
three B cell tumors.

\begin{center}
\begin{tabular}[8]{|l|l|l|}
B cell lineage tumor&surface IgM&secreted IgM\\
\hline
B1&+&-\\
B2&+&+\\
B3&-&+
\end{tabular}
\end{center}

\begin{exercise}[Northern Blot Analysis]
Determine with lane of the Northern blot corresponds with each of the
B cell lineage tumors.

\begin{center}
\begin{tabular}[5]{|l|l|}
TUMOR&NORTHERN BLOT LANE\\
B1&\sp\\
B2&\sp\\ 
B3&\sp\\
\end{tabular}
\end{center}
\end{exercise}

\end{document}