Chaffinch meiotic chromosomes in the lampbrush form: cytogenetic and molecular study

Alsu Saifitdinova, Svetlana Derjusheva and Elena Gaginskaya
Biological Institute of Saint-Petersburg University, Saint-Petersburg, 198504 RUSSIA
Fringilla coelebs L. is a numerous representative of Fringillidae (Aves: Passeriformes) having a pronounced sexual dimorphism and a wide distribution in the world. Its karyotype is shown to be typical of passerine birds (Piccini & Stella, 1970; De Boer, 1984; Derjusheva et al., 2001). In our Laboratory, the chaffinch is used as a model object to study different aspects of avian oogenesis, lampbrush chromosome structure and functions, avian genome organization.
Chaffinch LBCs
Enlarge in new window (75K)

Chaffinch chromosome in the lampbrush phase have typical lampbrush chromosome traits. They are of a really giant size, revealing a very high level of transcription on lateral loops and being decorated with a number of special landmarks. The most striking feature of chaffinch lampbrush chromosomes is occurence of specific protein bodies on each of bivalents (arrows).

Drawing of chaffinch macrobivalients in LBC stage
Enlarge in new window (21K)

Schematic drawing of chaffinch lampbrush macrobivalents. Characteristic pattern of lateral loops and other landmarks allows to recognize each individual bivalent.

So-called protein bodies were shown to be a characteristic peculiarity of lampbrush chromosomes in various groups of birds except representatives of Galliformes (Gaginskaya, 1972; Gaginskaya & Gruzova, 1975; Solovei et al., 1996). They are round bodies of variable size attached to the chromosome axis at heterochromatic regions. Their centromeric position was proved for pigeon lampbrush chromosomes but the nature of these structures has not yet been determined. In appearance, they resemble the sphere organelles of amphibian lampbrush chromosomes which were found to be involved in the dynamics of transcriptional and splicing factors and to be a kind of Cajal bodies. However, up to now it was not known whether the two structures shared any functional homology.

Here we present new data on centromeric location of protein bodies in the chaffinch. The protein bodies were shown to have no relation to Cajal bodies since no main components of the Cajal bodies were found in protein bodies by immunocytochemical techniques.

AntigensCBsSn-BPBsAntibodies used for PBsResources
Sm epitope++Y12J. Gall
TMG cap++K121J. Gall
Spl. factorSC35+/–+ASC35J. Gall
Fibrillarin+17C12J. Gall
Coilin+Rabbit serumJ. Gall
B23++
two — a few
small spots
3C9Artemenko et al., 2000
Phosphoryl. CTD of RNA-pol II–(+)++H5J. Gall (Tsvetkov)
Unphosphoryl. CTD if RNA-pol II++SWG16J. Gall (Tsvetkov)

A list of antibodies applied to chaffinch lampbrush chromosomes to compare the chaffinch protein bodies with both Cajal bodies and B-snurposomes from amphibian oocytes. The results show significant differences in molecular marker components between the structures and protein bodies.

Immunostaining with Y12
Enlarge in new window (76K)

Immunostaining of chaffinch lampbrush chromosomes with Y12 mAb against Sm-epitope of snRNPs. Arrows indicate unstained protein bodies. FITC/PI.

Evidence for protein bodies association with the sites of FCP centromeric repeat localization

Colocalization of PBs and FCP by FISH
Enlarge in new window (43K)

Lampbrush bivalent I of chaffinch. A — phase contrast; B — co-localization of protein bodies and FCP as revealed by FISH with FCP probe, Cy3 detection

Colocalization of PBs and FCP by PRINS
Enlarge in new window (33K)

Lampbrush bivalent I of chaffinch. A — phase contrast; B — co-localization of protein bodies and FCP as revealed by PRINS with FCP specific primers, Cy3 detection

In some birds, in addition to the centromeric protein bodies small ones were revealed in non-centromeric regions of certain lampbrush chromosome. We made microdissections of individual protein bodies of both types from bivalents with following PCR analysis using FCP specific primers. It was shown that all protein bodies contain FCP sequence and was concluded that all types of protein bodies are formed on lampbrush chromosomes in association with the FCP repeat.

Additional PBs
Enlarge in new window (72K)
Dissected sample PCR products EF
Enlarge in new window (34K)

A, B — lampbrush bivalent I of chaffinch. A — phase contrast, arrowheads indicate centromeric protein bodies, arrow indicates the additional protein body. B — the same bivalent after microdissection. CMA/PI. C — electrophoresis of dissected sample PCR products: 1 — negative control (the material lacked any protein body), 2 — molecular mass marker (lambda/HindIII), 3 — chromosome fragment with centromeric protein body, 4 — chromosome fragment with additional protein body.

Evidence for DNA transcription within the protein bodies

EM-ultrastructure of PBs
Enlarge in new window (187K)

Ultrastructure of protein bodies. A, B, C - ultrathin sections across the protein body. C - detection of DNA (arrows) and RNA (arrowheads) by Bernhardís technique

Transcription of FCP in PBs
Enlarge in new window (27K)

Detection of satDNA transcription in protein bodies by FISH according to DNA/RNA hybridization protocol with FCP probe. Cy3/DAPI.

Immunofluorescence of RNA-pol II
Enlarge in new window (23K)

Detection of RNA-polymerase II on lampbrush chromosomes by staining with 8WG16 mAB. FITC/PI.

References

De Boer L. (1984) Genetica 65: 3–38
Gaginskaya E. (1972) Tsitologiia 14: 568–578
Gaginskaya E. & Gruzova M. (1975) Tsitologiia 17: 1132–1137
Piccini E. & Stella M. (1970) Caryologia 23: 189–202
Solovei I. et al. (1996) Chromosome Res. 4: 588–603

© Laboratory of Chromosome Structure and Function, 2001