the RF-2 gene required for normal translation of
amber-recoded genes. Open reading frames in
the LA domain are almost entirely devoid of
in-frame amber codons and instead rely nearly
exclusively on canonical glutamine codons to
encode for glutamine (Fig. 4A). In contrast, the
high-amber (HA) domain with frequent in-frame
amber codons contains genes often associated
with late stages of phage infection, such as packaging and assembly components (e.g., predicted
tail fiber protein, minor tail protein, tail tape measure protein, or tail-associated lysozyme) (Fig. 4A).
This distinct codon utilization, combined with
the presence of RF-2 and a Gln-tRNACUA in the
amber-recoded phage, suggests that the amber-recoded phage actively interferes with the translation of its presumed opal-recoded host through
a proposed mode of phage-host antagonism (Fig.
4B). In this model, upon initial phage infection
abundant host-derived RF-1 (the releasing factor
that terminates peptide chain elongation at amber
codons) interferes with the translation of amber-containing phage HA domain genes, so they are
initially not expressed. In contrast, critical amber-free phage LA domain genes can be normally translated. Next, phage-derived expression of RF-2
increasingly interferes with translation of opal-recoded host genes. Last, the simultaneous depletion in host-derived RF-1 and the increasing
availability of phage-derived Gln-tRNACUA enable
the efficient production of assembly and packaging
proteins from the phage HA-domain. Although
direct in vivo observations of such processes remain
to be established, this evidence supports a mechanism of phage-host antagonism in which the
host’s viability is undermined by the phage through
the targeted codon-based disruption of the translation of the host’s genetic code.
This survey of environmental sequence data
revealed the abundance and diversity of stop
codon reassignments in prokaryotes and phages.
Several lines of evidence suggest that phages are
not obligated to adapt to the codon usage of their
hosts and that phages can exploit differences in
codon usage to manipulate their hosts. Recently,
genomically recoded organisms were created
in an attempt to isolate the organism’s genetic
information from horizontal transfer to natural
organisms and viruses (9). The diversity and abundance of recoding among uncultured environmental microbes and their phages suggests that
even synthetic genomically recoded organisms
(9) may not be immune to the exchange of genetic
information with microbes and phages that populate many ecosystems.
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We thank the DOE JGI production sequencing, IMG, and Genomes
OnLine Database teams for their support and J. Kim, A. Tadmor,
and A. Nord for reviewing the manuscript. The work conducted by
the DOE JGI was supported in part by the Office of Science of DOE
under contract DE-AC02-05CH11231. Supporting data can be
accessed through www.jgi.doe.gov and can be downloaded from
Materials and Methods
Figs. S1 to S12
Tables S1 to S11
10 January 2014; accepted 23 April 2014
Activating hotspot L205R
mutation in PRKACA and adrenal
Yanan Cao,1 Minghui He,2 Zhibo Gao,2 Ying Peng,1 Yanli Li,1 Lin Li,2 Weiwei Zhou,1
Xiangchun Li,2 Xu Zhong,1 Yiming Lei,2 Tingwei Su,1 Hang Wang,2 Yiran Jiang,1
Lin Yang,2 Wei Wei,1 Xu Yang,2 Xiuli Jiang,1 Li Liu,2 Juan He,1 Junna Ye,1 Qing Wei,3
Yingrui Li,2 Weiqing Wang,1† Jun Wang,2,4,5,6,7† Guang Ning1,8†
Adrenal Cushing’s syndrome is caused by excess production of glucocorticoid from adrenocortical
tumors and hyperplasias, which leads to metabolic disorders. We performed whole-exome
sequencing of 49 blood-tumor pairs and RNA sequencing of 44 tumors from cortisol-producing
adrenocortical adenomas (ACAs), adrenocorticotropic hormone–independent macronodular
adrenocortical hyperplasias (AIMAHs), and adrenocortical oncocytomas (ADOs). We identified
a hotspot in the PRKACA gene with a L205R mutation in 69.2% (27 out of 39) of ACAs and
validated in 65.5% of a total of 87 ACAs. Our data revealed that the activating L205R mutation,
which locates in the P+1 loop of the protein kinase A (PKA) catalytic subunit, promoted PKA
substrate phosphorylation and target gene expression. Moreover, we discovered the recurrently
mutated gene DOT1L in AIMAHs and CLASP2 in ADOs. Collectively, these data highlight
potentially functional mutated genes in adrenal Cushing’s syndrome.
Cushing’s syndrome is caused by excessive glucocorticoid production, which may lead to a series of metabolic disorders such as obesity, glucose intolerance, and hyper- tension. Adrenal Cushing’s syndrome results from autonomous production of cortisol
from adrenocortical tumors (ACTs), which are
most common in adult females. Cortisol-producing
ACTs include benign adrenocortical adenoma (ACA),
malignant adrenocortical carcinoma (ACC), and
rare forms of bilateral adrenal hyperplasia (BAH)
and adrenocortical oncocytoma (ADO) (1). BAHs
consist of macronodular and micronodular hyperplasias, such as adrenocorticotropin-independent
macronodular adrenocortical hyperplasia (AIMAH)
and primary pigmented nodular adrenocortical
Several genetic alterations have been described
in inherited or sporadic BAHs. The inactivating
mutations in PRKAR1A [cyclic adenosine mono-
phosphate (cAMP)–dependent protein kinase
regulatory subunit type Ia] are dominant in
1Shanghai Clinical Center for Endocrine and Metabolic
Diseases, Shanghai Key Laboratory for Endocrine Tumors,
Rui-Jin Hospital, Shanghai Jiao-Tong University School of
Medicine, Shanghai, China. 2BGI-Shanghai, BGI-Shenzhen,
Shenzhen, China. 3Department of Pathology, Rui-Jin Hospital,
Shanghai Jiao-Tong University School of Medicine, Shanghai,
China. 4Department of Biology, University of Copenhagen,
Copenhagen, Denmark. 5King Abdulaziz University, Jeddah,
Saudi Arabia. 6Macau University of Science and Technology,
Macau, China. 7Department of Medicine, University of
Hong Kong, Hong Kong. 8Laboratory of Endocrinology and
Metabolism, Institute of Health Sciences, Shanghai Institutes
for Biological Sciences (SIBS), Chinese Academy of
Sciences (CAS), and Shanghai Jiao Tong University School
of Medicine (SJTUSM), Shanghai, China.
*These authors contributed equally to this work. †Corresponding
author. E-mail: email@example.com (G.N.); wangj@
genomics.org.cn (J. W.); firstname.lastname@example.org (W. W.)