Comprehensive FISH Probe Design Tool Applied to Imaging Human Immunoglobulin Class Switch Recombination | PLOS One

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Figure 1.

Human immunoglobulin heavy chain constant region genes and specific FISH probes.
(a) The relative positions of the nine immunoglobulin heavy chain constant genes, to , in tandem array on chromosome 14 are illustrated by blue, green and red horizontal double bars. They are located near the telomere of human chromosome 14, downstream of the heavy chain variable, diversity and joining (VDJ) genes. Switching from the production of IgM to IgE, is caused by class switch recombination. It involves the juxtaposition of the replacement heavy chain constant region gene to the VDJ genes through the deletion of the intervening chromosomal DNA that forms a switch circle. (b–c) The genes ( to ) are superimposed on the background of colors representing the chromosomal DNA similarity identified by webFISH. The white background highlights unique sequences, which lack similarity to the rest of the genome. The gray background denotes sequences similar more than one genome sequences. The cyan background marks a locally repetitive subset of the gray similar sequences. These sequences share similarity exclusively within the chromosomal DNA segment in (c), but not with any other part of the genome. (b)-specific (blue horizontal double bars, A–F) and -specific (red horizontal double bars, G–K) single-copy FISH probes were identified within the contiguous stretches of unique (white background) sequences exceeding 4 kilobases in length. (c)-specific repetitive FISH probes (green horizontal double bars, x&y) were designed to bind only locally repeating sequences (including the adjacent green filled bars) but not any other part of the genome. The combined length of the -specific FISH probes is 14.3 kb but overall it binds 37.0 kb of sequence.

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Figure 1.

Human immunoglobulin heavy chain constant region genes and specific FISH probes.
(a) The relative positions of the nine immunoglobulin heavy chain constant genes, to , in tandem array on chromosome 14 are illustrated by blue, green and red horizontal double bars. They are located near the telomere of human chromosome 14, downstream of the heavy chain variable, diversity and joining (VDJ) genes. Switching from the production of IgM to IgE, is caused by class switch recombination. It involves the juxtaposition of the replacement heavy chain constant region gene to the VDJ genes through the deletion of the intervening chromosomal DNA that forms a switch circle. (b–c) The genes ( to ) are superimposed on the background of colors representing the chromosomal DNA similarity identified by webFISH. The white background highlights unique sequences, which lack similarity to the rest of the genome. The gray background denotes sequences similar more than one genome sequences. The cyan background marks a locally repetitive subset of the gray similar sequences. These sequences share similarity exclusively within the chromosomal DNA segment in (c), but not with any other part of the genome. (b)-specific (blue horizontal double bars, A–F) and -specific (red horizontal double bars, G–K) single-copy FISH probes were identified within the contiguous stretches of unique (white background) sequences exceeding 4 kilobases in length. (c)-specific repetitive FISH probes (green horizontal double bars, x&y) were designed to bind only locally repeating sequences (including the adjacent green filled bars) but not any other part of the genome. The combined length of the -specific FISH probes is 14.3 kb but overall it binds 37.0 kb of sequence.

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Figure 2.

Class switch recombination states detected by FISH.
(a–g) IgM, IgG or IgE-expressing cells represented by combinations of “μ” (A–F, blue), “” (x&y, green) or “” (G–K, red) FISH probes. Gray background stains (a) DNA in a population of pure IgM-expressing cells or (b–g) the expressed immunoglobulin class in cells enriched for IgG or IgE expression. Dashed lines approximate the nuclear outline. Details about the applied image processing algorithm are presented in “Materials and Methods”. (g) Decomposition of panel (d) into raw images of individual spectral channels (raw 2D data for a–f are presented in Figure S1 and raw 3D data are available in Supplementary Software, https://github.com/webfish/). Scale bar, 2 µm. (h) Each of the four classes, IgM, IgG, IgG and IgE, was studied in one to four patients (1–4). From patient 4 only IgE cells were examined. The number of scrutinized cells from each patient and the patient identifiers are inside and below the graph bars, respectively.

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Figure 2.

Class switch recombination states detected by FISH.
(a–g) IgM, IgG or IgE-expressing cells represented by combinations of “μ” (A–F, blue), “” (x&y, green) or “” (G–K, red) FISH probes. Gray background stains (a) DNA in a population of pure IgM-expressing cells or (b–g) the expressed immunoglobulin class in cells enriched for IgG or IgE expression. Dashed lines approximate the nuclear outline. Details about the applied image processing algorithm are presented in “Materials and Methods”. (g) Decomposition of panel (d) into raw images of individual spectral channels (raw 2D data for a–f are presented in Figure S1 and raw 3D data are available in Supplementary Software, https://github.com/webfish/). Scale bar, 2 µm. (h) Each of the four classes, IgM, IgG, IgG and IgE, was studied in one to four patients (1–4). From patient 4 only IgE cells were examined. The number of scrutinized cells from each patient and the patient identifiers are inside and below the graph bars, respectively.

More »