Features of the Chicken Genome Relevant to the Study of Aging

The chicken karyotype consists of 39 pairs of chromosomes, which is typical of most avian species. The genome is organized as eight pairs of cytologically distinct macrochromosomes, the Z and W sex chromosomes and thirty pairs of small cytologically indistinguishable microchromosomes (ICSGS, 2004). As in other vertebrates, chicken telomeres consist of a highly conserved hexanucleotide repeat, 5' TTAGGG(n) 3'. The cytogenetic features of the telomere repeat were first described in chicken by Nanda and Schmid (1994). Molecular features of telomeric DNA in the chicken genome were described in 2000 (Delany et al.). Although the avian genome is one-third the size of the human genome (1.25 pg versus 3 pg/haploid cell), the amount of telomeric DNA sequence is five to ten times more abundant in birds than in humans (Delany et al. , 2000; Nanda et al., 2002). Higher telomere repeat content in the chicken is likely due to the high number of chromosome ends (2n = 78 or 156 chromosome termini), the load of interstitial telomeric DNA and the presence of an unusual category of ultra-long telomeric arrays (see Figure 29.1).

Telomeric DNA in the chicken can be categorized into three main array size classes. Class I telomere repeats are 0.5-10 kb in length and exhibit discrete and genotype-specific banding patterns. Class I repeats are interstitially located and show no evidence of telomere shortening. Class II repeats are 10-40 kb and appear on Southern blots as the typical overlapping smear of TRFs; Class II arrays show evidence of terminal location based on digestion by Bal 31 and exhibit division-dependent shortening in somatic tissues. Class III telomeres are hundreds of kilobases in size and range to 3 megabases. Shortening of these arrays has not been established because of the inability to resolve changes of 100s of nucleotides (typical telomere erosion) in the context

Figure 29.1. Image of pulse-field gel showing chicken Class I, II and III telomere arrays. Class II arrays are analyzed for telomere shortening.

of 100s to 1000s of kilobases of the Class III arrays (Delany et al., 2000). In order to resolve Class III arrays on a gel, special pulse field gel electrophoresis parameters are required (Delany et al., 2000).

Not all avian species exhibit the Class III arrays (Delany et al., 2000; Nanda et al., 2002). Current models suggest that the Class III arrays of the chicken map to a subset of microchromosomes, perhaps serving to protect these small genetic elements from erosion and/or contributing to high microchromosome recombination rates (Delany et al., 2000; Delany et al., 2003). It is important to note that the existence of megabase telomere arrays in chicken does not diminish the power of the chicken as a model for division-dependent telomere shortening as it appears to be the shortest telomere or the unprotected telomere which triggers genome instability (Hemann et al., 2001; Karlseder et al., 2002).

Telomerase activity and telomere-shortening profiles in avian cells in vivo and in vitro mirror what is observed in human cells. Telomerase activity is develop-mentally regulated in vivo with high levels of telomerase in early-stage chicken embryos (preblastula through neurula) and during organogenesis all organs surveyed up to 10 days of embryonation (E10) followed by down-regulation for most somatic tissues. Constitutive telomerase activity continues for "renewable" tissues, including intestine, spleen, and organs or cells of the reproductive system. An average decrease of 3.2 kb in telomere length was observed from the early embryo to the adult (Taylor and Delany, 2000). In vitro observations include absence of telomerase from nontrans-formed primary cells (CEFs) contrasted with telomerase activity in cultured blastodermal cells, cES cells and in every transformed avian cell type surveyed to date (Table 29.3 ).

As measured by mean TRF, the in vitro rate of telomere shortening observed in Class II arrays in CEFs is approximately 50 bp of telomeric DNA per population doubling. Yet calculation of percent telomeric DNA at representative passages revealed that an average of 63% of the telomeric DNA was eroded in CEFs by senescence. The greatest loss of telomeric DNA occurred precipitously in later passages. These data suggest two mechanisms of telomere shortening: (1) telomere attrition due to the end-replication problem and (2) catastrophic erosion preceding culture arrest (Swanberg and Delany, 2003).

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