Here we show that the paralogue of this protein, WFIKKN1, also binds to both myostatin and myostatin propeptide and that both WFIKKN1 and WFIKKN2 bind GDF11, the growth and differentiation factor most closely related to myostatin, with high affinity. Structure-function studies on WFIKKN1 have revealed that the follistatin domain is primarily responsible for the binding of mature growth factor, whereas the NTR domain contributes most significantly to the interaction with myostatin propeptide. Analysis of the evolutionary histories of WFIKKN1/WFIKKN2 and GDF8/GDF11 proteins indicates that the functional association of an ancestral WFIKKN protein with an ancestor of GDF8/11 may date back to cephalochordates/urochordates. Although duplication of the corresponding genes gave rise to WFIKKN1/WFIKKN2 and GDF8/GDF11 in early vertebrates, the data presented here suggest that there is significant functional overlap of the paralogous proteins.By using sensitive homology search and gene finding programs, we have previously identified two closely related multidomain proteins of unknown function: WFIKKN and WFIKKNRP (1, 2). Both proteins (recently renamed as WFIKKN1 and WFIKKN2) contain a WAP domain, a follistatin/Kazal domain, an immunoglobulin domain, two Kunitztype protease inhibitor domains, and an NTR domain. Because WAP-, Kazal-, and Kunitz-type protease inhibitor modules are frequently found in serine protease inhibitor proteins (3) and the inhibitory N-terminal domain of tissue inhibitors of metalloproteases belongs to the NTR domain family (4), we have suggested that WFIKKNs may be multivalent protease inhibitors that may control the action of different proteases.Although the second Kunitz-type protease inhibitor domain of human WFIKKN1 protein was indeed found to inhibit trypsin (and only trypsin) out of a panel of trypsin-related serine proteases (5), structural and evolutionary analyses suggest that trypsin may not be the prime target of this Kunitz domain (6).Studies on the expression characteristics of WFIKKN1 and WFIKKN2 did not provide obvious clues as to the biological function of these proteins as both genes were expressed in multiple tissues (1, 2). The WFIKKN1 gene was found to be expressed (in order of decreasing intensity) in pancreas, thymus, liver, kidney, lung, and testis with practically no expression in brain, heart, skeletal muscle, ovary, small intestine, colon, leukocyte, spleen, and prostate. The WFIKKN2 gene is expressed (in order of decreasing intensity) in ovary, testis, pancreas, brain, and lung but not in heart, liver, placenta, small intestine, colon, leukocyte, spleen and prostate. Thus the main difference between the two genes is that the WFIKKN2 gene is expressed in brain but not in liver, whereas the reverse is true for the WFIKKN1 gene.In the case of fetal tissues, the WFIKKN1 gene is expressed at the highest level in the lung with weaker expression in skeletal muscle and liver, whereas the WFIKKN2 gene is expressed primarily in fetal brain, skeletal muscle, thymus, and kidney. Thus, ...

Human DNA sequence variation data are useful for studying the origin, evolution, and demographic history of modern humans and the mechanisms of maintenance of genetic variability in human populations, and for detecting linkage association of disease. Here, we report worldwide variation data from a Ϸ10-kilobase noncoding autosomal region. We identified 75 variant sites in 64 humans (128 sequences) and 463 variant sites among the human, chimpanzee, and orangutan sequences. Statistical tests suggested that the region is selectively neutral. The average nucleotide diversity () across the region was 0.088% among all of the human sequences obtained, 0.085% among African sequences, and 0.082% among non-African sequences, supporting the view of a low nucleotide diversity (Ϸ0.1%) in humans. The comparable value in nonAfricans to that in Africans indicates no severe bottleneck during the evolution of modern non-Africans; however, the possibility of a mild bottleneck cannot be excluded because non-Africans showed considerably fewer variants than Africans. The present and two previous large data sets all show a strong excess of low frequency variants in comparison to that expected from an equilibrium population, indicating a relatively recent population expansion. The mutation rate was estimated to be 1.15 ؋ 10 ؊9 per nucleotide per year. Estimates of the long-term effective population size Ne by various statistical methods were similar to those in other studies. The age of the most recent common ancestor was estimated to be Ϸ1.29 million years ago among all of the sequences obtained and Ϸ634,000 years ago among the non-African sequences, providing the first evidence from a noncoding autosomal region for ancient human histories, even among non-Africans.human origins ͉ nucleotide diversity ͉ rare variants ͉ population expansion