Origin of Human Chromosome 2 Revisited

9indian Academyof Sciences

Origin of human chromosome 2 revisited

REA V. SAMONTE*, K. H. RAMESH t and RAM S. VERMA Division of Genetics, The Long Island College Hospital, SUNY Health Science Center at Brooklyn, NY 11201, USA

Abstract

Similarities in chromosome banding patterns and homologies in DNA sequence between chromosomes of the great apes and humans have suggested that human chromosome 2 originated through the fusion of two ancestral ape chromosomes. A lot of work has been directed at understanding the nature and mechanism of this fusion. The recent availability of the human chromosome-2-specific alpha satellite DNA probe D2Z and the human chromosome-2p-specific subtelomeric DNA probe D2S445 prompted us to attempt cross-hybridization with chromosomes of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orangutan (Pongopygmaeus) to search for equivalent locations in the great apes and to comment on the origin of human chromosome 2. The probes gave different results. No hybridization to the chromosome-2-specific alpha satellite DNA probe was observed on the presumed homologous great ape chromosomes using both high-stringency and low- stringency post-hybridization washes, whereas the subtelomeric-DNA probe specific for chromosome 2p hybridized to telomeric sites of the short arm of chromosome 12 of all ttu'ee great apes. These observations suggest an evolutionary difference in the number of alpha satellite DNA repeat units in the equivalent ape chromosomes presumably involved in the chromosome fusion. Nevertheless, complete conservation of DNA sequence of the subtelomeric repeat sequence D2S445 in the ape chromosomes is demonstrated.

[Samonte R. V., Ramesh K. H. and Verma R. S. 1998 Origin of human cln'omosome2 revisited. Z Genet. 77, 41-44]

Introduction has been characterized (Waye and Willard 1987; Blackburn 1991). Both centromere and telomere repeat units are Comparison of karyotypes among the hominoid species distinct characteristics of primate chromosomes (Meyne helps in reconstructing the number and types of chromo- et al. 1989; Adinolfi 1992), and have been investigated in some rearrangements that presumably occurred since these chimpanzee chromosomes (Luke and Verma 1993, 1995). IL species were separated by evolution. The chromosome is the aim of this investigation to search for the equivalent number of the great ape species was established as 48 locations of the human chromosome 2 centromere and short- (Young et al. 1960; Chiarelli 1961; Hamerton et aI. 1961), arm telomere in the ape chromosomes to provide additional while that of humans is 46. The difference in chromosome evidence concerning the origin of human chromosome 2. number could be explained by the joining of two different acrocentric chromosomes in a common m~cestor by fusion to form the large submetacentric human chromosome 2 Materials and methods (Dutrillaux 1975). In recent years the structure of centromeres and telomeres of several human chromosomes Metaphase spreads were prepared from fibroblast cell lines of chimpanzee (GMO 3448A, Pan troglodytes, PTR), gorilla (AGO 5251A, Gorilla gorilla, GGO) and orangutan (GMO 6213, Pongo pygmaeus, PPY) obtained from the * Present address: Natural Sciences Research Institute, University of Coriell Institute for Medical Research, Camden, NJ, the Philippines, Diliman, Quezon City, Philippines t Present address: Department of Pathology, Albeit Einstein USA. Human (HSA) chromosomes were prepared from College of Medicine, Bronx, NY 10461, USA PHA-stimulated lymphocyte cultures obtained from healthy

Keywords. human chromosome 2; chimpanzee; gorilla; orangutan; alpha satellite DNA; subtelomeric repeat.

Journal of Genetics, Vol. 77, No. 1, April 1998 41 Rea V. Samome et al.

individuals. Harvesting of cell cultures was done using standard protocols (Verrna and Babu 1995). Fluorescence in situ hybridization (FISH) was performed according to Lichter et al. (1988). Briefly, chromosome preparations were dehydrated through an ethanol series and denatured in 70% formamide in 2 x SSC (pH 7.0) at 72~ The DNA probes used were biotin-labelled human chromosome-2-specific alpha satellite DNA probe D2Z and digoxigenin-labelled human tel 2p DNA probe D2S445 (Oncor, Gaithersburg, MD, USA). Overnight hybridization was performed at 37~ For the slide containing the alpha satellite DNA probe, both high-stringency and low-stringency post-hybridization washes were performed using 0.25 x and 2.0 x SSPE (pH 7.0) respectively at 70~ and detection was performed using fluorescein-labelled avidin. For the slide containing the tel 2p DNA probe, post- hybridization wash was done using 2 x SSPE (pH 7.0) at 70~ and detection was performed using fluorescein- labelled anti-digoxigenin. Chromosomes were counter- stained with propidium iodide/antifade and observed under fluorescence optics. Photographs were taken using Ekta- chrome EPH (Kodak) film and digital images were obtained using a cooled CCD camera (Oncor).

Results and discussion PTR12 G(3012 PPY12

p 2~ Similarities in chromosome banding patterns and homo- p2~ ~p22 p22 logies in DNA sequence between great ape and human p2~ plG plz.plS '1 ~ P~ plZ.p~6 chromosomes suggest that human chromosome 2 originated p12 lip plz ~ ~ by fusion of two ancestral ape chromosomes (Dutrillaux 1975; Yunis and Prakash 1982; ISCN 1985; Wieuberg q12 et aL 1990, 1994; Luke and Verma 1992). Genetic evidence suggests that this event must have occurred only a few i q Z~ q 24 million years ago (Goldstein et al. 1995; Horai et al. 1995). llq Although the precise nature of this fusion is unknown, molecular data have shown that telomeric DNA is present in q3~ human chromosomal band 2q13, suggesting that telomeres, the extreme ends of chromosomes, may have been involved HSA2 PIR13 GG011 PPY11 in this fusion (Allshire et aI. 1988; Wells et al. 1990; Ijdo C et al. 1991). Normally, telomeres form a dynamic buffer Figure 1. (A) Human chromosome 2 (HSA 2) centromere show- against loss of internal sequences and prevent chromosomes ing positive signal when hybridized with human chromosome-2- from fusing (Zakian 1989). It has been demonstrated that specific alphoid DNA probe D2Z (Oncor, Gaithersburg). The centromeres of chimpanzee chromosomes 12, 13 (PTR 12, 13), band 2q21 corresponds to the site of an inactive centromere gorilla chromosomes 12, 11 (GGO 12, 11)and orangutan chromo- of a chromosome of chimpanzee. Non-centromeric alpha somes 12, 11 (PPY 12, 11) did not show positive signals when satellite DNA sequences have been identified in band hybridized with the D2Z human DNA probe. (B) Telomeric region [email protected]@2.1, further supporting the derivation of these of the short arm of chromosome HSA2 showing positive signal sequences from ancestral centromeres (Avarello et al. 1992; when hybridized with human telomere 2p DNA probe D2S445. The telomeric regions in the short arms of chromosomes PTR 12, Baldini et al. 1993). The alpha satellite DNA is a class of GGO 12 and PPY 12 also show positive signals when hybridized repetitive DNA that makes up 3-5% of each primate with the human telomere 2p DNA probe. (C) Idiogram of chromo- chromosome and is localized to all centromeric regions somes HSA 2, PTR 12 and 13, GGO 12 and 11, and PPY 12 and 11 (Adinolfi 1992). (ISCN 1985). Telomeres are important for chromosome stability and replication. Such functions are mediated by highly con- et al. 1990). Telomeric and subtelomeric DNA sequences served short tandem repeats (TzAG3)~ at the very ends of have been characterized in a number of human chromo- telomeres in all vertebrates (Moyzis et al. 1988; Meyne somes and have been shown to consist of repetitive elements

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Received 14 July 1997

44 Journal of Genetics, Vol, 77, No. 1, April 1998