Nucleotide sequence was obtained for a region of 7,099 bp spanning the nifU, nifS, nifV, niJW, nijZ, and nijM genes from Azotobacter chroococcum. Chromosomal mutations constructed at several sites within the locus confirmed a requirement for this region for expression of the molybdenum nitrogenase in this organism. The genes are tightly clustered and ordered as in Kkebsiella pneumoniae except for two additional open reading frames (ORFs) between nitfV and niJW. The arrangement of genes in A. chroococcum closely matches that described for Azotobacter vinelandii. The polypeptide encoded by ORF4 immediately downstream from nifV is 41% identical over 186 amino acids to the product of the cysE gene from Escherichia coli, which encodes serine acetyltransferase (SAT), a key enzyme in cysteine biosynthesis. Plasmids which potentially express ORF4 complemented E. coli JM39, a cysteine auxotroph which lacks SAT. SAT activity was detected in crude extracts of one such complemented strain. A strain of A. chroococcum carrying a chromosomal disruption of ORF4 grew normally with ammonium as the N source but more slowly than the parental strain when N2 was the sole N source. These data suggest that ORF4 encodes a nif-specific SAT required for optimizing expression of nitrogenase activity. ORF4 was assigned the name nijP. nijP may be required to boost rates of synthesis or intracellular concentrations of cysteine or methionine. Sequence identity between nitfV and leuA gene products suggests that nifV may catalyze a condensation reaction analogous to that carried out by isopropylmalate synthase (LEUA) but in which acetyl coenzyme and a-ketoglutarate are substrates for the formation of homocitrate, the proposed product of NIFV activity.Members of the genus Azotobacter are free-living, obligately aerobic, heterotrophic diazotrophs found in soil and freshwater environments. They are especially interesting from the point of view of N2 fixation because, depending on the species, they possess two or three genetically distinct nitrogenase systems whose expression is determined by metal availability (8). The genetics of N2 fixation in these organisms is complicated not only by their possession of alternative nitrogenases but also by their tolerance of 02 when fixing N2 (61). Our studies have concentrated on Azotobacter chroococcum MCD1, a derivative of ATCC 4412. This organism makes a typical molybdenum-dependent nitrogenase when molybdenum is available (77) which is replaced in the absence of molybdenum and presence of vanadium by a vanadium-dependent nitrogenase (60).Many of the genes required for N2 fixation were first defined in the facultative anaerobe Klebsiella pneumoniae M5A1, which possesses a molybdenum nitrogenase system only and fixes N2 in anaerobic or, at best, microaerobic environments. In this organism, a maximum of 20 specific nif genes are required for N2 fixation. They are organized as a contiguous cluster spanning approximately 24 kb containing seven or eight transcriptional units (2,16,57). The nifH, nifD, and nifK g...

Three variants were misnamed in the text and mislabeled in Figure 2. The relevant sentence has been changed to read, ''The three de novo CHD8 mutations observed in CM1 include two stop-gain mutations (GenBank: NM_001170629.2; c.4414C>T [p.Arg1472*] and c.4514G>A [p.Trp1505*]) and a splice donor site mutation (c.2907þ1G>T [p.?]).

Chiari I malformation (CM1), the displacement of the cerebellum through the foramen magnum into the spinal canal, is one of the most common pediatric neurological conditions. Individuals with CM1 can present with neurological symptoms, including severe headaches and sensory or motor deficits, often as a consequence of brainstem compression or syringomyelia (SM). We conducted whole-exome sequencing (WES) on 668 CM1 probands and 232 family members and performed gene-burden and de novo enrichment analyses. A significant enrichment of rare and de novo non-synonymous variants in chromodomain (CHD) genes was observed among individuals with CM1 (combined p ¼ 2.4 3 10 À10 ), including 3 de novo loss-of-function variants in CHD8 (LOF enrichment p ¼ 1.9 3 10 À10 ) and a significant burden of rare transmitted variants in CHD3 (p ¼ 1.8 3 10 À6 ). Overall, individuals with CM1 were found to have significantly increased head circumference (p ¼ 2.6 3 10 À9 ), with many harboring CHD rare variants having macrocephaly. Finally, haploinsufficiency for chd8 in zebrafish led to macrocephaly and posterior hindbrain displacement reminiscent of CM1. These results implicate chromodomain genes and excessive brain growth in CM1 pathogenesis.

Cerebrospinal fluid (CSF) is essential for the development and function of the central nervous system (CNS). However, the brain and its interstitium have largely been thought of as a single entity through which CSF circulates, and it is not known whether specific cell populations within the CNS preferentially interact with the CSF. Here, we develop a technique for CSF tracking, gold nanoparticle-enhanced X-ray microtomography, to achieve micrometer-scale resolution visualization of CSF circulation patterns during development. Using this method and subsequent histological analysis in rodents, we identify previously uncharacterized CSF pathways from the subarachnoid space (particularly the basal cisterns) that mediate CSF-parenchymal interactions involving 24 functional-anatomic cell groupings in the brain and spinal cord. CSF distribution to these areas is largely restricted to early development and is altered in posthemorrhagic hydrocephalus. Our study also presents particle size-dependent CSF circulation patterns through the CNS including interaction between neurons and small CSF tracers, but not large CSF tracers. These findings have implications for understanding the biological basis of normal brain development and the pathogenesis of a broad range of disease states, including hydrocephalus.

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