Table 1. Summary and characterization of knockout strains identified as AOX deficient.
| Knockout Library Grid Number | Mutant Class | Panel of Figure 2 Showing Western | NCU Numbera | FGSC Number | Known or Predicted Protein or Genea (Domain Identified) | Growth Defectb | Growth on Antimycin A for48 hrc | Conidiation Defectd |
|---|---|---|---|---|---|---|---|---|
| 1C3e | 1 | na | 03938.5 | 11227 | aod-5 | 0 | ||
| 23H2 | 1 | A | 08887.5 | 15957 | Hypothetical protein (major facilitator superfamily) | 0 | ||
| 40E6f | 1 | A | 05600.5 | 13805 | Ubiquinone biosynthesis protein (ABC1 family) | 0 | ||
| 12096.5 | Aspartyl aminopeptidase (glycosyl hydrolase family) | |||||||
| 41G6 | 1 | A | 03589.5 | 13923 | Hypothetical protein | Slight | 0 | Slight |
| 47H10 | 1 | A | 00778.5 | 16938 | sed5 vesicle protein (transmembrane adaptor Erv26) | Severe | 0 | Severe |
| 52D8 | 1 | B | 07281.5 | 14469 | Glucose-6-phosphate isomerase (phosphoglucose isomerase) | Severe | 0 | Slight |
| 83H3 | 1 | A | 08365.5 | 18277 | RNA polymerase II mediator complex component Med8 | Slight | 0 | Severe |
| 88H8 | 1 | A | 01542.5 | 19221 | Hypothetical protein (HbrB like domain) | 0 | ||
| 96H9e | 1 | na | 07953.5 | 18947 | aod-1 | |||
| 97B1e | 1 | na | 03352.5 | 19465 | aod-2 | |||
| 100B5 | 1 | A | 08158.5 | 19644 | Dual-specificity phosphatase | Slight | 0 | Slight |
| 1G9 | 2 | D | 00157.5 | 11281 | COP 9 signalosome-1, csn-1 | Slight | 0 | Slight |
| 1G11 | 2 | D | 00467.5 | 11283 | csn-5 | Slight | 0 | Slight |
| 2A3 | 2 | D | 08741.5 | 11299 | Striatin pro 11 (WD domain, G-beta repeat) | 0.7 | Severe | |
| 6A1 | 2 | A | 06799.5 | 11001 | Fungal specific transcription factor, vad-5 | 0 | ||
| 6F2 | 2 | B | 09739.5 | 11062 | Hypothetical protein, ada-7 (Zn(2)-Cys(6) binuclear cluster) | 0.7 | Severe | |
| 7H4 | 2 | B | 03875.5 | 11780 | Chromatin remodeling complex ATPase chain ISW1 (SLIDE, HAND, helicase conserved domain, SNF2 family, type III restriction enzyme, DEAD/DEAH box helicase, class II histone deactylase complex subunits 2 and 3) | 0 | Severe | |
| 12C8 | 2 | D | 01266.5 | 12022 | Phosphoinositide-specific phospholipase C | 0.6 | ||
| 13G11 | 2 | D | 04566.5 | 12420 | Protein kinase SNF1 | 0 | Slight | |
| 14A5 | 2 | D | 03727.5 | 12091 | Hyphal anastomosis-2, ham-2 | 0.4 | Severe | |
| 28C5 | 2 | C | 00007.5 | 16098 | pH response regulator palH, rim-21 | 1.0 | Slight | |
| 28D10 | 2 | C | 00593.5 | 16116 | COP9 signalosome-2, csn-2 | Slight | 0.2 | Slight |
| 30C2 | 2 | B | 06199.5 | 13193 | RNA binding protein Jsn1 (RNA recognition motif) | 1.2 | Severe | |
| 44H2 | 2 | B | 04826.5 | 16834 | Hypothetical protein | 0 | Severe | |
| 45C8 | 2 | D | 03035.5 | 13973 | Hypothetical protein | 0.3 | Slight | |
| 50G11 | 2 | A | 04607.5 | 14316 | Hypothetical protein | 0.3 | Slight | |
| 81E9 | 2 | E | 06084.5 | 20114 | Hypothetical protein (Vps51/Vps67) | 1.6 | Severe | |
| 83E1 | 2 | B | 07881.5 | 18239 | Hypothetical protein | 2.7 | Severe | |
| 85H5 | 2 | E | 09803.5 | 18375 | Thioredoxin (glutaredoxin homolog) | Slight | 0 | Slight |
| 96C10 | 2 | B | 04245.5 | 18888 | Translocase of outer mitochondrial membrane 70, tom70 | 0 | Severe | |
| 96G8g | 2 | E | 06727.5 | 18934 | Spermidine 3, spe-3 (Spermidine synthase) | 2.3 | ||
| 2C4 | 3 | D | 03894.5 | 11324 | Serine/threonine protein kinase ste-20 | 0.2 | ||
| 4B3 | 3 | E | 04062.5 | 11473 | Peroxisome biogenesis factor 20 | 1.1 | ||
| 4F11 | 3 | G | 08480.5 | 11671 | Hsf-type DNA-binding domain-containing protein | 0 | Severe | |
| 9E5 | 3 | E | 08565.5 | 11945 | Hypothetical protein (NACHT domain) | 0.4 | ||
| 10B5 | 3 | F | 09212.5 | 11545 | Serine/threonine protein kinase | 1.7 | ||
| 10B6 | 3 | F | 06563.5 | 11546 | Serine/threonine protein phosphatase PP2A catalytic subunit (calcineurin-like phosphoesterase) | Slight | 0.5 | Severe |
| 10B8 | 3 | H | 07489.5 | 11548 | Phosphatase-Z-like-1, pzl-1 (calcineurin-like phosphoesterase) | 0.4 | Severe | |
| 10C6 | 3 | H | 03853.5 | 11559 | Peptidyl-prolyl cis-trans isomerase (tetratricopeptide repeat) | 2.7 | Slight | |
| 13C11 | 3 | F | 06493.5 | 12370 | Guanine nucleotide-binding protein alpha-1 (ADP ribosylation factor) | 3.3 | ||
| 28B10 | 3 | G | 08137.5 | 16091 | Hypothetical protein | 0 | Slight | |
| 30F5 | 3 | C | 01471.5 | 13235 | Nuclear protein SNF4 (CBS domain) | 0 | ||
| 32A11 | 3 | C | 01955.5 | 13376 | Autophagocytosis protein Aut1 | 1.9 | Slight | |
| 42A2 | 3 | D | 01276.5 | 16557 | N-acetyltransferase 5 | Slight | 0.5 | Slight |
| FR47-like protein | ||||||||
| 48E6g | 3 | C | 04771.5 | 16944 | Fructosyl-amino acid oxidase (FAD dependent oxidoreductase) | 0.5 | Slight | |
| 51F9 | 3 | C | 09530.5 | 14398 | Hypothetical protein | 1.4 | ||
| 52F6 | 3 | C | 08067.5 | 14491 | Osmosensor protein (SH3 domain) | 3.5 | ||
| 63E6 | 3 | H | 03802.5 | 17474 | Trimethyllysine dioxygenase (taurine catabolism dioxygenase) | 2.6 | Slight | |
| 82B6 | 3 | E | 07112.5 | 20169 | Hypothetical protein (pyridine nucleotide-disulphide oxidoreductase) | 1.5 | ||
| 83F11 | 3 | F | 01511.5 | 18261 | Urease accessory protein ureG | Severe | 0.5 | Severe |
| 92B11 | 3 | G | 07579.5 | 19411 | Hypothetical protein (SCA7) | 0.2 | Severe | |
| 93F6 | 3 | G | 03708.5 | 18633 | Hypothetical protein (putative methyl transferase) | 1.8 | ||
| 94D6g | 3 | G | 00712.5 | 18704 | 3-hydroxy-3-methylglutaryl-coenzyme A reductase (sterol sensing domain, patched family) | 2.6 | ||
| 97D9 | 3 | G | 00360.5 | 19497 | NAD-dependent epimerase/dehydratase | 0 | Slight | |
| 98H11 | 3 | G | 10058.5 | 18976 | Phosphoglucomutase 2 | 1.1 | ||
| 99C10 | 3 | E | 09686.5 | 20379 | Clock-controlled gene-8, ccg-8 (transcription factor opl1) | 0 | ||
| 100C8 | 3 | C | 09208.5 | 19659 | Transcription factor SPT8 (WD domain, G-beta repeat) | 0 | ||
| 100F2 | 3 | C | 09527.5 | 20459 | Hypothetical protein | 2.0 | ||
| 101D3 | 3 | F | 09560.5 | 21068 | Superoxide dismutase | 0 | ||
| 103G4 | 3 | F | 09553.5 | 21156 | 3-hydroxybutyryl CoA dehydrogenase | 2.2 | ||
| 103G10g | 3 | H | 08132.5 | 21162 | Alpha-1,3-glucan synthase, Ags2 | Slight | 0.5 | |
| 104D1 | 3 | F | 02973.5 | 21213 | Mitochondrial carrier protein | 0.1 | ||
| 104E9 | 3 | C | 01553.5 | 21233 | Para-hydroxybenzoate-polyprenyltransferase, Coq2 | 0 | ||
| 108C9 | 3 | H | 02979.5 | 21401 | AMP deaminase | 1.8 | Slight | |
| 108G10g | 3 | H | 08279.5 | 21450 | Hypothetical protein | 1.1 |
Information from N. crassa database BROAD Institute (http://www.broadinstitute.org/annotation/genome/neurospora/MultiHome.html).
Growth rates were measured by inoculating conidiaspores at the center of Petri dishes (standard 100 mm × 15 mm size) containing Vogel’s medium. Plates were incubated at 30°. The radius of mycelial extension from the inoculation point was measured (in cm) and compared with the wild-type control strain that covers the surface of the plate (radius of growth from the inoculation point of 4.2 cm) in 48 hr. Strains with a “slight” growth defect were defined as having a growth radius of 1.5 to 4.1 cm in 48 hr. A “severe” growth defect was defined as having a growth radius of less than 1.5 cm in 48 hr.
Same as b, but the medium contained Antimycin A. Measurements of growth (in cm) were taken after 48 hr. The control strain grew 1.7 cm in 48 hr on average under these conditions.
Formation of asexual conidiaspores was considered slightly defective if conidiation on slants containing 3 mL of Vogel’s medium in a 1 cm × 10-cm test tube was similar to the control but took longer than the control strain. The defect was considered severe if very few conidia formed, even after extended times.
The aod-1 (Li et al. 1996), aod-2, and aod-5 (Descheneau et al. 2005) genes were all identified during the screen and have been described in detail previously.
Original predicted open reading frame was knocked out but is now known to be divided into two predicted genes.
Strain maintained in the knockout library as a heterokaryon. This implies that the affected gene is essential for viability.