Figure 1.
The thylacine, Thylacinus cynocephalus.
(a) Young male thylacine in Hobart Zoo in 1928, photograph (Q4437). (b) One of the preserved pouch young specimens (head length 34 mm) from which DNA was extracted, from the Museum Victoria collection. (c-f) The skull of the thylacine (c,e) compared with that of the domestic dog Canis canis (d,f). The morphology of the head shows remarkable convergent evolution. However, there are some differences: in marsupials, the lacrymal extends outside the orbit and the angle of the dentary is medially inflected (c). The thylacine palatine has the vacuities characteristic of marsupial skulls (e). The teeth also show striking convergent evolution but the muzzle of the thylacine is quite narrow compared to that of the dog (e,f). Scale bar = 5cm.
Figure 2.
Analyses of the thylacine Col2a1 enhancer element.
(a) Sequence alignment of the thylacine PCR cloned Col2a1 enhancer. Identical alignment between species is shown by black boxes. The thylacine sequence was most similar to, but distinct from that of another marsupial, the tammar wallaby, Macropus eugenii. The black box outlines the minimal 18-bp element. Inverted repeats (arrows) surrounding the minimal element are shown with complementary nucleotides in red. The outer repeat is highly conserved between all mammals, but the inner repeat differs by 2 nucleotides in the thylacine. (b) Phylogenetic analysis of the thylacine Col2a1 enhancer. The thylacine sequence groups with the tammar wallaby, both of which are more similar to the human sequence than to mouse or rat. Numbers indicate bootstrap values based on 100 replicates.
Figure 3.
From extinction to gene expression.
Functional analysis of the thylacine non-coding DNA fragment. (a) Diagram of transgene construct. 4 copies of a 264-bp fragment containing the Thylacine Col2a1 enhancer (TcyCol2a1) region was ligated to the human b-globin minimal promoter (black box) and ligated to lacZpA. (b–e) X-gal stained 14.5 dpc TcyCol2a1-lacZpA transgenic mouse embryo showing varying levels of reporter gene expression within the developing cartilage (blue). (f) Non-transgenic littermate, negative control fetus. (g) Top panel; Magnified image of forelimb from fetus in (b) black line indicates the plane of section shown in (g) bottom panel. Bottom panel; Histological section of transgenic forelimb digit, showing lacZ-expressing chondrogenic tissue (blue) counterstained with eosin (pink).