The Fuzzy planar cell polarity protein (FUZ), necessary for primary cilium formation,is essential for pituitary development

The primary cilium is an essential organelle that is important for normal cell signalling during development and homeostasis but its role in pituitary development has not been reported. The primary cilium facilitates signal transduction for multiple pathways, the best-characterised being the SHH pathway, which is known to be necessary for correct pituitary gland development. FUZ is a planar cell polarity (PCP) effector that is essential for normal ciliogenesis, where the primary cilia of Fuz^−/−mutants are shorter or non-functional. FUZ is part of a group of proteins required for recruiting retrograde intraflagellar transport proteins to the base of the organelle. Previous work has reported ciliopathy phenotypes in Fuz^−/− homozygous null mouse mutants, including neural tube defects, craniofacial abnormalities, and polydactyly, alongside PCP defects including kinked/curly tails and heart defects. Interestingly, the pituitary gland was reported to be missing in Fuz^−/− mutants at 14.5 dpc but the mechanisms underlying this phenotype were not investigated. Here, we have analysed the pituitary development of Fuz^−/− mutants. Histological analyses reveal that Rathke's pouch (RP) is initially induced normally but is not specified and fails to express LHX3, resulting in hypoplasia and apoptosis. Characterisation of SHH signalling reveals reduced pathway activation in Fuz^−/− mutant relative to control embryos, leading to deficient specification of anterior pituitary fate. Analyses of the key developmental signals FGF8 and BMP4, which are influenced by SHH, reveal abnormal patterning in the ventral diencephalon, contributing further to abnormal RP development. Taken together, our analyses suggest that primary cilia are required for normal pituitary specification through SHH signalling.     

Chronic up‐regulation of sonic hedgehog has little effect on postnatal craniofacial morphology of euploid and trisomic mice

Background: In Ts65Dn, a mouse model of Down syndrome (DS), brain and craniofacial abnormalities that parallel those in people with DS are linked to an attenuated cellular response to sonic hedgehog (SHH) signaling. If a similarly reduced response to SHH occurs in all trisomic cells, then chronic up‐regulation of the pathway might have a positive effect on development in trisomic mice, resulting in amelioration of the craniofacial anomalies. Results: We crossed Ts65Dn with Ptch1^tm1Mps/+ mice and quantified the craniofacial morphology of Ts65Dn;Ptch^+/? offspring to assess whether a chronic up‐regulation of the SHH pathway rescued DS‐related anomalies. Ts65Dn;Ptch1^+/? mice experience a chronic increase in SHH in SHH‐receptive cells due to haploinsufficiency of the pathway suppressor, Ptch1. Chronic up‐regulation had minimal effect on craniofacial shape and did not correct facial abnormalities in Ts65Dn;Ptch^+/? mice. We further compared effects of this chronic up‐regulation of SHH with acute pathway stimulation in mice treated on the day of birth with a SHH pathway agonist, SAG. We found that SHH affects facial morphology differently based on chronic vs. acute postnatal pathway up‐regulation. Conclusions: Our findings have implications for understanding the function of SHH in craniofacial development and for the potential use of SHH‐based agonists to treat DS‐related abnormalities. Developmental Dynamics 245:114–122, 2016. © 2015 Wiley Periodicals, Inc.     

Family-based study of association between ENPP1 genetic variants and craniofacial morphology

Background: Human craniofacial morphology is characterized by considerable diversity among individuals. The ENPP1 gene is essential for bone physiology. However, the potential effects of its genetic variants on head size phenotypes have not yet been studied.

Aim: The aim of this research was to investigate the association of polymorphisms in the ENPP1 locus with normal variability of craniofacial phenotypes.

Subjects and methods: Fourteen SNPs and 13 haplotypes in the ENPP1 locus were tested for association with six head size traits in 1042 Western Eurasian individuals.

Results: The most significant and consistent association was observed between upper facial height and the polymorphisms located near the promoter region and upstream from ENPP1 gene (p = 0.00009), which remained significant after adjustment for multiple testing. Additionally, association signals were detected between head breadths and lower face height, and markers residing in or close to the promoter and 3^′ untranslated regions of the ENPP1 gene (p < 0.05).

Conclusions: The findings obtained in this study suggest that the upstream, promoter and 3^′ untranslated regions in the ENPP1 locus harbor genetic variants affecting different aspects of craniofacial morphology. Further research is required to validate the relevancy of the potentially functional ENPP1 regions to normal and pathologic craniofacial growth.     

PAC1 deficiency attenuates progression of atherosclerosis in ApoE deficient mice under cholesterol-enriched diet

The neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP) is vasoactive and cytoprotective and exerts immunoregulatory functions throughout the nervous, neuroendocrine cardiovascular and immune systems in health and disease. PACAP mainly acts through PAC1 receptor signaling in neuronal communication, but the role of PAC1 in immune regulation of atherosclerosis is not known. Here, we generated PAC1^−/−/ApoE^−/− mice to test, whether PAC1^−/− influences plasma cholesterol-/triglyceride levels and/or atherogenesis in the brachiocephalic trunk (BT) seen in ApoE^−/− mice, under standard chow (SC) or cholesterol-enriched diet (CED). Furthermore, the effect of PAC1^−/−, on inflammatory, autophagy-, apoptosis- and necroptosis-relevant proteins in atherosclerotic plaques was determined. In plaques of PAC1^−/−/ApoE^−/− mice fed a SC, the immunoreactivity for apoptotic, autophagic, necroptotic and proinflammatory proteins was increased, however, proliferation was unaffected. Interestingly, without affecting hyperlipidemia, PAC1^−/− in ApoE^−/− mice remarkably reduced CED-induced lumen stenosis seen in ApoE^−/− mice. Thus, PAC1^−/− allows unchecked inflammation, necroptosis and decreased proliferation during SC, apparently priming the BT to develop reduced atheroma under subsequent CED. Remarkably, no differences in inflammation/necroptosis signatures in the atheroma under CED between PAC1^−/−/ApoE^−/− and ApoE^−/− mice were observed. These data indicate that selective PAC1 antagonists should offer potential as a novel class of atheroprotective therapeutics, especially during hypercholesterolemia.     

Cross-presentation of dead-cell-associated antigens by DNGR-1+ dendritic cells contributes to chronic allograft rejection in mice

The purpose of this study was to elucidate whether DC NK lectin group receptor-1 (DNGR-1)-dependent cross-presentation of dead-cell-associated antigens occurs after transplantation and contributes to CD8⁺ T cell responses, chronic allograft rejection (CAR), and fibrosis. BALB/c or C57BL/6 hearts were heterotopically transplanted into WT, Clec9a^−/−, or Batf3^−/− recipient C57BL/6 mice. Allografts were analyzed for cell infiltration, CD8⁺ T cell activation, fibrogenesis, and CAR using immunohistochemistry, Western blot, qRT²-PCR, and flow cytometry. Allografts displayed infiltration by recipient DNGR-1⁺ DCs, signs of CAR, and fibrosis. Allografts in Clec9a^−/− recipients showed reduced CAR (p < 0.0001), fibrosis (P = 0.0137), CD8⁺ cell infiltration (P < 0.0001), and effector cytokine levels compared to WT recipients. Batf3-deficiency greatly reduced DNGR-1⁺ DC-infiltration, CAR (P < 0.0001), and fibrosis (P = 0.0382). CD8 cells infiltrating allografts of cytochrome C treated recipients, showed reduced production of CD8 effector cytokines (P < 0.05). Further, alloreactive CD8⁺ T cell response in indirect pathway IFN-γ ELISPOT was reduced in Clec9a^−/− recipient mice (P = 0.0283). Blockade of DNGR-1 by antibody, similar to genetic elimination of the receptor, reduced CAR (P = 0.0003), fibrosis (P = 0.0273), infiltration of CD8⁺ cells (p = 0.0006), and effector cytokine levels. DNGR-1-dependent alloantigen cross-presentation by DNGR-1⁺ DCs induces alloreactive CD8⁺ cells that induce CAR and fibrosis. Antibody against DNGR-1 can block this process and prevent CAR and fibrosis.     

The NLRP3 inflammasome contributes to host protection during Sporothrix schenckii infection

Sporotrichosis is a mycosis caused by fungi from the Sporothrix schenckii species complex, whose prototypical member is Sporothrix schenckii sensu stricto. Pattern recognition receptors (PRRs) recognize and respond to pathogen‐associated molecular patterns (PAMPs) and shape the following adaptive immune response. A family of PRRs most frequently associated with fungal recognition is the nucleotide‐binding oligomerization domain‐like receptor (NLR). After PAMP recognition, NLR family pyrin domain‐containing 3 (NLRP3) binds to apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC) and caspase‐1 to form the NLRP3 inflammasome. When activated, this complex promotes the maturation of the pro‐inflammatory cytokines interleukin‐1β (IL‐1β) and IL‐18 and cell death through pyroptosis. In this study, we aimed to evaluate the importance of the NLRP3 inflammasome in the outcome of S. schenckii infection using the following three different knockout (KO) mice: NLRP3^−/−, ASC^−/− and caspase‐1^−/−. All KO mice were more susceptible to infection than the wild‐type, suggesting that NLRP3‐triggered responses contribute to host protection during S. schenckii infection. Furthermore, the NLRP3 inflammasome appeared to be critical for the ex vivo release of IL‐1β, IL‐18 and IL‐17 but not interferon‐γ. Additionally, a role for the inflammasome in shaping the adaptive immune response was suggested by the lower frequencies of type 17 helper T (Th17) cells and Th1/Th17 but not Th1 cells in S. schenckii‐infected KO mice. Overall, our results indicate that the NLRP3 inflammasome links the innate recognition of S. schenckii to the adaptive immune response, so contributing to protection against this infection.     

Oestrogen modulates cardiac ischaemic remodelling through oestrogen receptor-specific mechanisms

Aim: Observational and clinical studies suggest different responses upon sex hormone replacement therapy in ischaemic heart disease. Few studies, however, have examined the impact of oestrogen receptor‐dependent mechanisms on the extent of injury after myocardial infarction (MI). Therefore, we set out to evaluate the effect of oestrogen (E2) replacement on infarct size and remodelling, and the respective role of the oestrogen receptors (ER)α and ‐β in this process, using ERα‐ and ERβ‐deficient mice. Methods: Wild type (WT) (ERα^+/+ and ERβ^+/+), ERα‐deficient (ERα^−/−) and ERβ‐deficient (ERβ^−/−) mice were ovariectomized and subsequently supplemented with E2 or placebo using subcutaneous 60‐day release pellets. MI was induced by left coronary artery ligation. Two weeks following MI, haemodynamic function was assessed and infarct size was determined. Results: There was no significant difference in infarct size between E2‐ or placebo‐treated WT (ERα^+/+ and ERβ^+/+) mice. Surprisingly, E2 treatment did result in smaller infarct sizes in ERα^−/− mice, but increased the infarct size in ERβ^−/− mice. Increase of the left ventricular mass post‐MI was significantly larger in the E2‐treated ERα^−/− animals compared with placebo‐treated animals. E2 treatment also significantly increased post‐MI mortality in ERα^+/+, ERβ^+/+ and ERα^−/− animals, but not in ERβ^−/− mice. Conclusions: Although E2 modulates the infarct size in ERα^−/−, it also appears to be responsible for the higher mortality following MI. ERβ appears to be the receptor involved in the modulating effects of E2 in the infarcted heart.     

Dok adaptors play anti‐inflammatory roles in pulmonary homeostasis

Asthma is a chronic inflammatory disease of the lung with airflow obstruction and bronchospasm, characterized by pulmonary eosinophilia, airway remodeling, increased airway hyperresponsiveness to environmental stimuli, and excessive Th2‐type cytokine production. Recent studies indicate that crosstalk between the innate and adaptive immune systems is crucial for this disease. We and others have showed that the Dok (downstream of tyrosine kinases) family adaptors, Dok‐1, Dok‐2, and Dok‐3, play essential roles in negative regulation of a wide variety of signaling pathways in both innate and adaptive immunities. Here, histopathology and bronchoalveolar lavage fluid (BALF) cellularity showed spontaneous pulmonary inflammation in Dok‐1^−/−Dok‐2^−/−Dok‐3^−/− (TKO) mice, but not in Dok‐1^−/−Dok‐2^−/− or Dok‐3^−/− mice, with hallmarks of asthma, including eosinophilia, goblet cell hyperplasia, and subepithelial fibrosis. Consistently, TKO mice, but not the other mutants, showed increased airway hyperresponsiveness to methacholine inhalation. In addition, Th2‐type cytokine concentrations in BALF were increased in TKO mice. These findings provide strong evidence that Dok‐1, Dok‐2, and Dok‐3 cooperatively play critical anti‐inflammatory roles in lung homeostasis.     

Three-dimensional form and function of the nasal cavity and nasopharynx in humans and chimpanzees

The facial differences between recent Pan troglodytes and Homo sapiens can be used as a proxy for the reduction of facial prognathism that happened during evolutionary transition between Australopithecines and early Homo. The projecting nasal morphology of Homo has been considered both a passive consequence of anatomical reorganization related to brain and integrated craniofacial evolution as well as an adaptation related to air-conditioning during physiological and behavioral shifts in human evolution. Yet, previous research suggested impaired air-conditioning in Homo challenging respiratory adaptations based on computational fluid dynamics (CFD) and airflow simulations. Here we improved CFD model at the inflow region and also carried out three-dimensional (3D) geometric morphometrics to address the hypothesis of impaired air-conditioning in humans and species differences in airway shape. With the new CFD model we simulated pressure, velocity, and temperature changes in airflow of six adult humans and six chimpanzees and analyzed 164 semi-landmarks of 10 humans and 10 chimpanzees for 3D size and shape comparisons. Our finding shows significantly different internal 3D nasal airways. Also, species means of pressure, velocity, and temperature differed statistically significantly. However, form-related differences in temperature exchanges seem subtle and may question adaptive disadvantages. We rather support a hypothesis of craniofacial changes in the Australopithecus-Homo transition that are related to brain evolution and craniofacial integration with facial and nasal modifications that contribute to maintain respiratory adaptations related to air conditioning.     

Cell-division-cycle defects associated with fission yeast pre-mRNA splicing mutants

We have isolated six new pre-mRNA splicing mutants (prp) from a collection of temperature-sensitive (ts^–) Schizosaccharomyces pombe strains. The prp mutants are defective in the splicing of both messenger RNA and U6 small nuclear RNA precursors. A single recessive mutation is responsible for both the ts ^– growth and the splicing phenotypes in each of the prp mutants. The six prp mutations are unlinked and fall into separate complementation groups. Two are allelic with the previously described prp3 and prp4 mutations; the remaining four define the new alleles prp5-1, prp6-1, prp7-1, and prp9-1. The six mutants exhibit three splicing phenotypes: accumulation of unspliced precursor at the restrictive but not at the permissive temperature; accumulation of unspliced precursor at both the permissive and restrictive temperatures; and accumulation of unspliced precursor, the intron-exon lariat intermediate, and the intron lariat final product. In addition to their aberrant splicing phenotypes, the prp5-1 and prp6-1 mutants express classical cell-division-cycle defects, while prp7-1 exhibits an unusual hyphal morphology. These results suggest a connection between pre-mRNA splicing and the control of cell division in fission yeast. Received: 1 June / 10 July 1998     

Morphological alterations of the reticular thalamic nucleus in Engrailed-2 knockout mice

The reticular thalamic nucleus (Rt) is a sheet of neurons that surrounds the dorsal thalamus laterally, along its dorso-ventral and rostro-caudal axes. It consists of inhibitory neurons releasing gamma-aminobutyric acid (GABA). This nucleus participates in the circuitry between the thalamus and the cerebral cortex, and its impairment is associated with neuro-psychiatric disorders. In this study, we investigated the Rt anatomy of Engrailed-2 knockout mice (En2^−/−), a mouse model of autism spectrum disorder (ASD), using parvalbumin as an immunohistochemical marker. We compared 4- and 6-week-old wild type (WT) and En2^−/− mice using various morphometric parameters: cell area, shape factor, circularity and cell density. Significant differences were present in 6-week-old male mice with different genetic background (WT vs. En2^−/−): the Rt neurons of En2^−/− mice showed a bigger cell area, shape factor and circularity when compared with WT. Age (4 weeks vs. 6 weeks) influenced the shape factor of WT females, the circularity and cell density of En2^−/− males, and the shape factor and circularity of En2^−/− females. Gender affected cell density in 4-week-old WT mice, shape factor and cellularity of 6-week-old WT mice, and cell area, shape factor and cell density of En2^−/− at 6 weeks. Intrasubject (left–right) asymmetry of Rt was never observed. These results show for the first time that sex- and age-related changes occur in the Rt GABAergic neurons of the En2^−/− ASD mouse model.     

Role of somite patterning in the formation of Weberian apparatus and pleural rib in zebrafish

In the vertebrate body, a metameric structure is present along the anterior–posterior axis. Zebrafish tbx6^−/− larvae, in which somite boundaries do not form during embryogenesis, were shown to exhibit abnormal skeletal morphology such as rib, neural arch and hemal arch. In this study, we investigated the role of somite patterning in the formation of anterior vertebrae and ribs in more detail. Using three-dimensional computed tomography scans, we found that anterior vertebrae including the Weberian apparatus were severely affected in tbx6^−/− larvae. In addition, pleural ribs of tbx6 mutants exhibited severe defects in the initial ossification, extension of ossification, and formation of parapophyses. Two-colour staining revealed that bifurcation of ribs was caused by fusion or branching of ribs in tbx6^−/−. The parapophyses in tbx6^−/− juvenile fish showed irregular positioning to centra and abnormal attachment to ribs. Furthermore, we found that the ossification of the distal portion of ribs proceeded along myotome boundaries even in irregularly positioned myotome boundaries. These results provide evidence of the contribution of somite patterning to the formation of the Weberian apparatus and rib in zebrafish.     

Histamine-1 receptor is not required as a downstream effector of orexin-2 receptor in maintenance of basal sleep/wake states

Aim: The effect of orexin on wakefulness has been suggested to be largely mediated by activation of histaminergic neurones in the tuberomammillary nucleus (TMN) via orexin receptor-2 (OX₂R). However, orexin receptors in other regions of the brain might also play important roles in maintenance of wakefulness. To dissect the role of the histaminergic system as a downstream mediator of the orexin system in the regulation of sleep/wake states without compensation by the orexin receptor-1 (OX₁R) mediated pathways, we analysed the phenotype of Histamine-1 receptor (H₁R) and OX₁R double-deficient (H₁R^−/−;OX₁R^−/−) mice. These mice lack OX₁R-mediated pathways in addition to deficiency of H₁R, which is thought to be the most important system in downstream of OX₂R. Methods: We used H₁R deficient (H₁R^−/−) mice, H₁R^−/−;OX₁R^−/− mice, OX₁R and OX₂R double-deficient (OX₁R^−/−;OX₂R^−/−) mice, and wild type controls. Rapid eye movement (REM) sleep, non-REM (NREM) sleep and awake states were determined by polygraphic electroencephalographic/electromyographic recording. Results: No abnormality in sleep/wake states was observed in H₁R^−/− mice, consistent with previous studies. H₁R^−/−;OX₁R^−/− mice also showed a sleep/wake phenotype comparable to that of wild type mice, while OX₁R^−/−; OX₂R^−/− mice showed severe fragmentation of sleep/wake states. Conclusion: Our observations showed that regulation of the sleep/wake states is completely achieved by OX₂R-expressing neurones without involving H₁R-mediated pathways. The maintenance of basal physiological sleep/wake states is fully achieved without both H₁ and OX₁ receptors. Downstream pathways of OX₂R other than the histaminergic system might play an important role in the maintenance of sleep/wake states.     

Chemerin partly mediates tumor-inhibitory effect of all-trans retinoic acid via CMKLR1-dependent natural killer cell recruitment

Down-regulated chemerin expression has been reported to correlate with poor prognosis of several types of cancer including melanoma. All-trans retinoic acid (atRA) is a potent inducer of chemerin, and we previously reported that atRA inhibited murine melanoma growth through enhancement of anti-tumor T-cell immunity. Here, we aimed to investigate whether loss of endogenous chemerin accelerated melanoma growth and whether chemerin was involved in the melanoma-inhibitory effect of atRA. We demonstrated that chemerin was constitutively expressed in the skin, which was down-regulated during murine melanoma growth. Rarres2^−/− mice, which are deficient in chemerin, exhibited aggravated tumor growth and impaired tumor-infiltrating natural killer (NK) cells that express CMKLR1, the functional receptor of chemerin. Topical treatment with atRA up-regulated skin chemerin expression, which was primarily derived from dermal cells. Moreover, atRA treatment significantly enhanced tumor-infiltrating NK cells, which was completely abrogated in Rarres2^−/− mice and Cmklr1^−/− mice, suggesting a dependency of NK cell recruitment on the chemerin–CMKLR1 axis in melanoma. Despite comparable melanoma growth detected in wild-type mice and Cmklr1^−/− mice, lack of CMKLR1 partially abrogated the melanoma-inhibitory effect of atRA. This may be due to the inability to enhance tumor-infiltrating NK cells in Cmklr1^−/− mice following atRA treatment. Collectively, our study suggests that down-regulation of chemerin could be a strategy used by cancers such as melanoma to impair anti-tumor NK cell immunity and identifies a new anti-tumor mechanism of atRA by up-regulating chemerin to enhance CMKLR1-dependent NK cell recruitment.     

Craniofacial Shape Variation in Twist1+/− Mutant Mice

Craniosynostosis (CS) is a relatively common birth defect resulting from the premature fusion of one or more cranial sutures. Human genetic studies have identified several genes in association with CS. One such gene that has been implicated in both syndromic (Saethre–Chotzen syndrome) and nonsyndromic forms of CS in humans is TWIST1. In this study, a heterozygous Twist1 knock out (Twist1^+/?) mouse model was used to study the craniofacial shape changes associated with the partial loss of function. A geometric morphometric approach was used to analyze landmark data derived from microcomputed tomography scans to compare craniofacial shape between 17 Twist1^+/? mice and 26 of their Twist1^+/+ (wild type) littermate controls at 15 days of age. The results show that despite the purported wide variation in synostotic severity, Twist1^+/? mice have a consistent pattern of craniofacial dysmorphology affecting all major regions of the skull. Similar to Saethre–Chotzen, the calvarium is acrocephalic and wide with an overall brachycephalic shape. Mutant mice also exhibited a shortened cranial base and a wider and shorted face, consistent with coronal CS associated phenotypes. The results suggest that these differences are at least partially the direct result of the Twist1 haploinsufficiency on the developing craniofacial skeleton. This study provides a quantitative phenotype complement to the developmental and molecular genetic research previously done on Twist1. These results can be used to generate further hypotheses about the effect of Twist1 and premature suture fusion on the entire craniofacial skeleton. Anat Rec, 297:826–833, 2014. © 2014 Wiley Periodicals, Inc.     

Scn3b knockout mice exhibit abnormal sino-atrial and cardiac conduction properties

Aim: In contrast to extensive reports on the roles of Na_v1.5 α-subunits, there have been few studies associating the β-subunits with cardiac arrhythmogenesis. We investigated the sino-atrial and conduction properties in the hearts of Scn3b^−/− mice. Methods: The following properties were compared in the hearts of wild-type (WT) and Scn3b^−/− mice: (1) mRNA expression levels of Scn3b, Scn1b and Scn5a in atrial tissue. (2) Expression of the β₃ protein in isolated cardiac myocytes. (3) Electrocardiographic recordings in intact anaesthetized preparations. (4) Bipolar electrogram recordings from the atria of spontaneously beating and electrically stimulated Langendorff-perfused hearts. Results: Scn3b mRNA was expressed in the atria of WT but not Scn3b^−/− hearts. This was in contrast to similar expression levels of Scn1b and Scn5a mRNA. Immunofluorescence experiments confirmed that the β₃ protein was expressed in WT and absent in Scn3b^−/− cardiac myocytes. Lead I electrocardiograms from Scn3b^−/− mice showed slower heart rates, longer P wave durations and prolonged PR intervals than WT hearts. Spontaneously beating Langendorff-perfused Scn3b^−/− hearts demonstrated both abnormal atrial electrophysiological properties and evidence of partial or complete dissociation of atrial and ventricular activity. Atrial burst pacing protocols induced atrial tachycardia and fibrillation in all Scn3b^−/− but hardly any WT hearts. Scn3b^−/− hearts also demonstrated significantly longer sinus node recovery times than WT hearts. Conclusion: These findings demonstrate, for the first time, that a deficiency in Scn3b results in significant atrial electrophysiological and intracardiac conduction abnormalities, complementing the changes in ventricular electrophysiology reported on an earlier occasion.     

Second-site antibiotic resistance mutations in the ribosomal region of yeast mitochondrial DNA

Summary A large proportion of the spontaneous erythromycin resistant mutants isolated from a strain carrying a previously-induced chloramphenicol resistance mutation at cap3 do not map at ery1, the locus most often associated with mitochondrial erythromycin resistance. Most of the new mutations are also nonallelic at spil, spi2, and other known antibiotic resistance loci within the 21S rRNA gene; they are allelic with each other and define the new locus, ery2. Induced second-site erythromycin resistant mutants from the cap ^r3 strain, as well as spontaneous or induced mutants from strains carrying a cap ^r 1 mutation, all tend to map at eryl. The cap ^r3 mutation is apparently necessary for the expression of erythromycin resistance resulting from a second mutation at ery2.