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A combined banding method that allows the reliable identification of chromosomes as well as differentiation of AT- and GC-rich heterochromatin

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Abstract

Сonstitutive heterochromatin areas are revealed by differential staining as C-positive chromosomal regions. These C-positive bands may greatly vary by location, size, and nucleotide composition. CBG-banding is the most commonly used method to detect structural heterochromatin in animals. The difficulty in identification of individual chromosomes represents an unresolved problem of this method as the body of the chromosome is stained uniformly and does not have banding pattern beyond C-bands. Here, we present the method that we called CDAG for sequential heterochromatin staining after differential GTG-banding. The method uses G-banding followed by heat denaturation in the presence of formamide with consecutive fluorochrome staining. The new technique is valid for the concurrent revealing of heterochromatin position due to differential banding of chromosomes and heterochromatin composition (AT-/GC-rich) in animal karyotyping.

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Abbreviations

αMEM:

Alpha minimum essential medium

ATrH:

AT-rich heterochromatin

CBG-banding:

C-bands revealed by barium hydroxide treatment followed by Giemsa staining

FISH:

Fluorescence in situ hybridization

CDAG:

Chromomycin A3-DAPI-after G-banding

Cen:

centromere

CMA3:

chromomycin A3

DABCO:

1,4-diazabicyclo[2,2,2]-octane

DAPI:

4′-6-diamidino-2-phenylindole

DIPI:

4-6-bis-(2-imidozolynyl-4H,5H)-2-phenylindole

DMSO:

Dimethyl sulfoxide

DPBS:

Dulbecco’s phosphate-buffered saline

EDTA:

Ethylenediaminetetraacetic acid

FBS:

Fetal bovine serum

GCrH:

GC-rich heterochromatin

GTG-banding (G-banding):

G-bands by trypsin followed by Giemsa staining

H:

heterochromatin

PBS:

Phosphate-buffered saline

RT:

room temperature

o/n:

overnight

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Acknowledgments

We would like to acknowledge Dr. David McMullen for providing the tissue for establishing the cell line used in this study. We acknowledge anonymous reviewers and the editor whose comments helped to improve the manuscript. We are thankful to Dr. Tatyana Kolesnikova for helpful discussion.

Funding

The work was supported by the Russian Science Foundation (RSF, 16-14-10009). The work on human karyotypes was supported by RFBR according to the research project No. 18-04-00826.

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Authors and Affiliations

  1. Institute of Molecular and Cellular Biology SB RAS, Novosibirsk, Russia

    Natalya A. Lemskaya, Anastasia I. Kulemzina, Violetta R. Beklemisheva, Larisa S. Biltueva, Anastasia A. Proskuryakova, Polina L. Perelman & Alexander S. Graphodatsky

  2. Novosibirsk State University, Novosibirsk, Russia

    Anastasia A. Proskuryakova, Polina L. Perelman & Alexander S. Graphodatsky

  3. Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health (NINDS/NIH), Bethesda, MD, USA

    John M. Hallenbeck

Authors
  1. Natalya A. Lemskaya
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  2. Anastasia I. Kulemzina
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  3. Violetta R. Beklemisheva
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  4. Larisa S. Biltueva
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  5. Anastasia A. Proskuryakova
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  6. John M. Hallenbeck
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  7. Polina L. Perelman
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  8. Alexander S. Graphodatsky
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Contributions

NAL conceived, designed, performed research, and analyzed data. NAL, AIK, VRB, LSB, AAP, PLP conducted experiments. ASG, VRB analyzed data. JMH provided the critical sample. NAL wrote the manuscript. PLP, VRB, LSB edited manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Natalya A. Lemskaya.

Additional information

Responsible Editor: Fengtang Yang

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Lemskaya, N.A., Kulemzina, A.I., Beklemisheva, V.R. et al. A combined banding method that allows the reliable identification of chromosomes as well as differentiation of AT- and GC-rich heterochromatin. Chromosome Res 26, 307–315 (2018). https://doi.org/10.1007/s10577-018-9589-9

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  • DOI: https://doi.org/10.1007/s10577-018-9589-9

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