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Augmented expression of a human gene for 8-oxoguanine DNA glycosylase (MutM) in B lymphocytes of the dark zone in lymph node germinal centers.

Kuo FC, Sklar J - J. Exp. Med. (1997)

Bottom Line: Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues.In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs.Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
B cells that mediate normal, T cell-dependent, humoral immune responses must first pass through germinal centers (GCs) within the cortex of antigenically stimulated lymph nodes. As they move through the dark zone and then the light zone in the GC, B cells are subjected to somatic hypermutation and switch recombination within their rearranged immunoglobulin genes and also participate in a number of other processes that control development into memory cells or cells specialized for antibody secretion. To investigate the molecular mechanisms that contribute to B cell development within GCs, we constructed a recombinant DNA library enriched for cDNAs derived from human genes expressed in B cells at this site. This library was found to contain a cDNA structurally and functionally related to genes in bacteria and yeast for the DNA repair enzyme 8-oxoguanine DNA glycosylase. Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues. In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs. Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

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Northern blot analysis of GCN6 and GCN5 expression. 10  μg of total RNA were loaded in each lane. (A) Autoradiogram of the blot  after hybridization with radiolabeled GCN5 cDNA. The three bands referred to in the text are labeled A, B, and C. (B) Photograph under ultraviolet light of the agarose gel used in preparation of the Northern blot after staining of the gel with ethidium bromide to indicate the amount and  integrity of the RNA loaded on the gel.
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Figure 1: Northern blot analysis of GCN6 and GCN5 expression. 10 μg of total RNA were loaded in each lane. (A) Autoradiogram of the blot after hybridization with radiolabeled GCN5 cDNA. The three bands referred to in the text are labeled A, B, and C. (B) Photograph under ultraviolet light of the agarose gel used in preparation of the Northern blot after staining of the gel with ethidium bromide to indicate the amount and integrity of the RNA loaded on the gel.

Mentions: Among the first 24 cDNAs used as probes, four showed some specificity for RNA in GC B cells. One of these, GCN5, detected two transcripts about 2.0 and 1.8 kb in size predominantly in GC B cells and tonsil, but to a much lesser extent in non-GC B cells, T cells, non–lymphoid human fetal tissues, or lymphoid tumor cell lines (Fig. 1, bands A and B). The larger of these transcripts (band B) is also present at low levels in the RNA from some of the non–lymphoid tissues; however, the smaller transcript (band A) is almost undetectable anywhere other than in the RNA of GC B cells and tonsil. Small amounts of a third transcript, ∼2.3 kb in size (band C), were also identified in RNA of liver, lung, and brain using the GCN5 cDNA as a probe.


Augmented expression of a human gene for 8-oxoguanine DNA glycosylase (MutM) in B lymphocytes of the dark zone in lymph node germinal centers.

Kuo FC, Sklar J - J. Exp. Med. (1997)

Northern blot analysis of GCN6 and GCN5 expression. 10  μg of total RNA were loaded in each lane. (A) Autoradiogram of the blot  after hybridization with radiolabeled GCN5 cDNA. The three bands referred to in the text are labeled A, B, and C. (B) Photograph under ultraviolet light of the agarose gel used in preparation of the Northern blot after staining of the gel with ethidium bromide to indicate the amount and  integrity of the RNA loaded on the gel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199107&req=5

Figure 1: Northern blot analysis of GCN6 and GCN5 expression. 10 μg of total RNA were loaded in each lane. (A) Autoradiogram of the blot after hybridization with radiolabeled GCN5 cDNA. The three bands referred to in the text are labeled A, B, and C. (B) Photograph under ultraviolet light of the agarose gel used in preparation of the Northern blot after staining of the gel with ethidium bromide to indicate the amount and integrity of the RNA loaded on the gel.
Mentions: Among the first 24 cDNAs used as probes, four showed some specificity for RNA in GC B cells. One of these, GCN5, detected two transcripts about 2.0 and 1.8 kb in size predominantly in GC B cells and tonsil, but to a much lesser extent in non-GC B cells, T cells, non–lymphoid human fetal tissues, or lymphoid tumor cell lines (Fig. 1, bands A and B). The larger of these transcripts (band B) is also present at low levels in the RNA from some of the non–lymphoid tissues; however, the smaller transcript (band A) is almost undetectable anywhere other than in the RNA of GC B cells and tonsil. Small amounts of a third transcript, ∼2.3 kb in size (band C), were also identified in RNA of liver, lung, and brain using the GCN5 cDNA as a probe.

Bottom Line: Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues.In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs.Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
B cells that mediate normal, T cell-dependent, humoral immune responses must first pass through germinal centers (GCs) within the cortex of antigenically stimulated lymph nodes. As they move through the dark zone and then the light zone in the GC, B cells are subjected to somatic hypermutation and switch recombination within their rearranged immunoglobulin genes and also participate in a number of other processes that control development into memory cells or cells specialized for antibody secretion. To investigate the molecular mechanisms that contribute to B cell development within GCs, we constructed a recombinant DNA library enriched for cDNAs derived from human genes expressed in B cells at this site. This library was found to contain a cDNA structurally and functionally related to genes in bacteria and yeast for the DNA repair enzyme 8-oxoguanine DNA glycosylase. Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues. In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs. Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

Show MeSH