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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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In situ hybridization on paraffin-embedded formalin-fixed sections of human tonsils. (A)A hematoxylin and eosin–stained section showing  the dark (DZ), light (LZ), and mantle (MZ) zones of a GC. (B–D) Sections hybridized with GCN6 cDNA into which digoxigenin nucleotides have been  incorporated, followed by detection with antidigoxigenin antibody conjugated to alkaline phosphatase and identification of the locations of bound antibody by deposition of dark blue phosphatase reaction product. The nuclei were counterstained with methyl green. (B) Sense strand probe of GCN6. (C  and D) Antisense probe of GCN6. (E and F) Antisense probe of GCN7, a cDNA derived from a gene apparently expressed primarily in mantle cells.  Original magnification was 40 for B, D, and F and 100 for A, C, and E.
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Figure 4: In situ hybridization on paraffin-embedded formalin-fixed sections of human tonsils. (A)A hematoxylin and eosin–stained section showing the dark (DZ), light (LZ), and mantle (MZ) zones of a GC. (B–D) Sections hybridized with GCN6 cDNA into which digoxigenin nucleotides have been incorporated, followed by detection with antidigoxigenin antibody conjugated to alkaline phosphatase and identification of the locations of bound antibody by deposition of dark blue phosphatase reaction product. The nuclei were counterstained with methyl green. (B) Sense strand probe of GCN6. (C and D) Antisense probe of GCN6. (E and F) Antisense probe of GCN7, a cDNA derived from a gene apparently expressed primarily in mantle cells. Original magnification was 40 for B, D, and F and 100 for A, C, and E.

Mentions: To analyze the distribution of expression of GCN6 DNA in lymph node tissue, in situ hybridization was performed with digoxigenin-labeled sense and antisense riboprobes generated from the 3′ end of GCN6 cDNA (Fig. 2, the region between S6 and A7). As seen in Fig. 4, C and D, hybridization with a GCN6-specific antisense probe was largely localized to the cells in the dark zone of GCs present within sections of tonsil. Examination under high magnification revealed that the cells showing the strongest hybridization had a nuclear morphology compatible with centroblasts. Weaker hybridization was seen in the light zone of GCs, but not in the B lymphocytes making up the mantle zone. In some sections, scattered large cells both outside the GC and between the light and dark zone of the GC displayed intense hybridization. The nature of these cells is not clear.


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)

In situ hybridization on paraffin-embedded formalin-fixed sections of human tonsils. (A)A hematoxylin and eosin–stained section showing  the dark (DZ), light (LZ), and mantle (MZ) zones of a GC. (B–D) Sections hybridized with GCN6 cDNA into which digoxigenin nucleotides have been  incorporated, followed by detection with antidigoxigenin antibody conjugated to alkaline phosphatase and identification of the locations of bound antibody by deposition of dark blue phosphatase reaction product. The nuclei were counterstained with methyl green. (B) Sense strand probe of GCN6. (C  and D) Antisense probe of GCN6. (E and F) Antisense probe of GCN7, a cDNA derived from a gene apparently expressed primarily in mantle cells.  Original magnification was 40 for B, D, and F and 100 for A, C, and E.
© Copyright Policy
Related In: Results  -  Collection

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Figure 4: In situ hybridization on paraffin-embedded formalin-fixed sections of human tonsils. (A)A hematoxylin and eosin–stained section showing the dark (DZ), light (LZ), and mantle (MZ) zones of a GC. (B–D) Sections hybridized with GCN6 cDNA into which digoxigenin nucleotides have been incorporated, followed by detection with antidigoxigenin antibody conjugated to alkaline phosphatase and identification of the locations of bound antibody by deposition of dark blue phosphatase reaction product. The nuclei were counterstained with methyl green. (B) Sense strand probe of GCN6. (C and D) Antisense probe of GCN6. (E and F) Antisense probe of GCN7, a cDNA derived from a gene apparently expressed primarily in mantle cells. Original magnification was 40 for B, D, and F and 100 for A, C, and E.
Mentions: To analyze the distribution of expression of GCN6 DNA in lymph node tissue, in situ hybridization was performed with digoxigenin-labeled sense and antisense riboprobes generated from the 3′ end of GCN6 cDNA (Fig. 2, the region between S6 and A7). As seen in Fig. 4, C and D, hybridization with a GCN6-specific antisense probe was largely localized to the cells in the dark zone of GCs present within sections of tonsil. Examination under high magnification revealed that the cells showing the strongest hybridization had a nuclear morphology compatible with centroblasts. Weaker hybridization was seen in the light zone of GCs, but not in the B lymphocytes making up the mantle zone. In some sections, scattered large cells both outside the GC and between the light and dark zone of the GC displayed intense hybridization. The nature of these cells is not clear.

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