Hsieh J et al. (2000) Annual Review of Genetics "Recognition and silencing of repeated DNA."

Fire A, Hsieh J

[Annual Review of Genetics, 2000]

Mechanisms for repetition of DNA pose both opportunities and challenges to a functional genome: opportunities for increasing gene expression by amplification of useful sequences, and challenges of controlling amplification by unwanted sequences such as transposons and viruses. Experiments in numerous organisms have suggested the likely existence of a general mechanism for recognition of repeated character in DNA. This review focuses (a) on the nature of these recognition mechanisms, and (b) on types of chromatin modification and gene silencing that are used to control repeated DNA.

Hsieh J et al. (1998) East Coast Worm Meeting "A C. Elegans RING Finger Protein That May Be Involved In Repeat-Dependent Silencing in Somatic Cells"

Hsieh J, Fire A

[East Coast Worm Meeting, 1998]

The activity patterns of somatic transgenes provide a means to study gene silencing in C. elegans. Transgene silencing phenomena have been observed in a variety of organisms, and are often associated with repeated structures. It has been found in C. elegans that certain transgenes present in long tandem arrays are silenced or show mosaic expression (i.e. many cells contain the transgene but lack detectable expression). Silencing can be relieved when the constructs are in a non-repetitive context: in F1 assays or imbedded in arrays with excess of genomic carrier DNA (1). In order to identify components of a silencing mechanism we have performed two types of mutagenesis screens. In one screen, we started with lines which contain a silenced gfp transgene and screened for a de-silenced phenotype. In another screen, we started with a line which contains a transgene that uniformly expresses in body wall muscle and screened for a reduction or a loss of expression. From the latter screen, we had identified a chromosomal mutation, tentatively designated tandem-array-modifier-1(tam-1), in which the expression of gfp was greatly reduced when compared to the original strain (thanks to Steve Kostas). Tam-1 has similar effects on a number of other reporter transgenes expressed in both muscle and non-muscle tissue. There is a temporal component to tam-1 effects: with all transgenes tested, expression is reduced in older tam-1 mutant animals with no obvious difference in embryos or early larval stages. Our tam-1 mutations are allelic to a set of mutations (called grt-1/slm-1) that have been characterized by Jing Liu and Chieh Chang in Paul Sternberg's lab (2, 3). They have shown that there is a subtle effect on several endogenous genes that are active in larval or adult stages. In molecular analysis, we have shown that tam-1 encodes a RING finger-containing protein of predicted molecular weight 107kd. RING finger containing-proteins have been implicated in a variety of processes such as DNA repair and recombination and transcriptional repression in development. Preliminary localization studies show that a tam-1::gfp fusion construct is localized to the nucleus of adult somatic cells. The gfp is undetectable in embryos and larval stages. We are in the process of making an antibody to TAM-1. The role of tam-1 is somewhat of a mystery. We are currently testing two possibilities: 1) TAM-1 plays a role in stimulating the activity of some genes in certain types of chromosomal contexts [e.g. repetitive arrays] 2) TAM-1 is a time-specific regulator of gene expression and plays a general role in gene activity in later life stages.

Hsieh J et al. (1997) International C. elegans Meeting "Now You See It, Now You Don't: Repeat-Induced Gene Silencing in C. elegans"

Fire A, Hsieh J

[International C. elegans Meeting, 1997]

Transgene activity patterns provide a means to study gene silencing in C. elegans. Transgene silencing phenomena have been observed in a variety of organisms, and are often associated with repeated structures. It has been found in C. elegans that certain transgenes present in long tandem arrays are silenced or show mosaic expression (i.e. many cells contain the transgene but lack detectable expression). Silencing can be relieved when the constructs are in a non-repetitive context: in F1 assays or imbedded in arrays with excess of genomic carrier DNA [Kelly, Xu, Fire wbg 14.1:64]. By mutagenizing lines with various silenced transgenes, and screening for a release of the silencing, one can potentially recover both cis- and trans-acting factors that affect this phenomenon. In one screen, we start with a tandem array composed of a marker plasmid (e.g. pRF4[rol-6]) with interspersed copies of a myo-2::gfp fusion construct that is driven by the unc-54 enhancer. The myo-2::gfp expresses strongly in pharyngeal muscles, and seems relatively insensitive to silencing. The unc-54 enhancer provides additional body wall muscle expression for this construct, but this expression is completely repressed in tandem array transgenes. Following mutagenesis, we have recovered a number of strains in which body wall expression has reappeared. Of six such stains characterized to date, all have resulted from alterations in the transgene. Examination of array structure by Southern analysis showed no gross differences between mutant and original lines. We are currently modifying these screens to allow recovery of chromosomal mutations in putative trans-acting factors. Another set of possible mutations would hyperactivate silencing mechanisms. In his mutagenesis of a non-mosaic myo-3::gfp line, Steve Kostas recovered four strains with weaker and incomplete expression of gfp in bodywall muscle. In at least two cases, the mosaic nature of transgene expression is due to a recessive chromosomal mutation. We are in the process of characterizing these mutations.

Hsieh J et al. (1999) Genes Dev "The RING finger/B-box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate context-dependent gene silencing in Caenorhabditis elegans."

Hsieh J, Sternberg PW, Chang C, Fire A, Kostas SA, Liu J

[Genes Dev, 1999]

Context-dependent gene silencing is used by many organisms to stably modulate gene activity for large chromosomal regions. We have used tandem array transgenes as a model substrate in a screen for Caenorhabditis elegans mutants that affect context-dependent gene silencing in somatic tissues. This screen yielded multiple alleles of a previously uncharacterized gene, designated tam-1 (for tandem-array-modifier). Loss-of-function mutations in tam-1 led to a dramatic reduction in the activity of numerous highly repeated transgenes. These effects were apparently context dependent, as nonrepetitive transgenes retained activity in a tam-1 mutant background. In addition to the dramatic alterations in transgene activity, tam-1 mutants showed modest alterations in expression of a subset of endogenous cellular genes. These effects include genetic interactions that place tam-1 into a group called the class B synMuv genes (for a Synthetic Multivulva phenotype); this family plays a negative role in the regulation of RAS pathway activity in C. elegans. Loss-of-function mutants in other members of the class-B synMuv family, including lin-35, which encodes a protein similar to the tumor suppressor Rb, exhibit a hypersilencing in somatic transgenes similar to that of tam-1 mutants. Molecular analysis reveals that tam-1 encodes a broadly expressed nuclear protein with RING finger and B-box motifs.

J Hsieh et al. (1999) International C. elegans Meeting "Context-dependent gene silencing in the soma of C. elegans"

J Hsieh, A Fire, S Kostas

[International C. elegans Meeting, 1999]

The activity patterns of somatic transgenes provide a means to study gene silencing in C. elegans . Transgene silencing phenomena have been observed in a variety of organisms, and are often associated with repetitive contexts. In both the soma and germline of C. elegans , certain transgenes present in long tandem arrays are silenced or show mosaic expression (i.e. many cells contain the transgene but lack detectable expression). Silencing can be relieved when the constructs are in a non-repetitive context: in F1 assays or imbedded in arrays with excess of genomic carrier DNA (1). In order to identify trans-acting factors that modulate the levels of context-dependent gene silencing, we have carried out a number of genetic screens. In one screen, we looked for a reduction of expression from a uniformly expressing bodywall muscle transgene. Three alleles of a single chromosomal gene were isolated. Mutations in this gene, designated tandem-array-modifier-1 ( tam-1 ) have similar effects on a number of different reporter transgenes with diverse expression patterns. Other tam-1 mutations were picked up independently in a screen by Liu, Chang, and Sternberg (2,3). The TAM-1 protein is nuclear and contains two cysteine-rich domains, a "RING" finger and a "B-box". Although the molecular function of these domains are unknown, roles in context-dependent gene expression have been suggested for several RING-finger proteins. One biological role of context-dependent silencing is suggested by the unexpected observation that tam-1 behaves as a class B SynMuv gene. The inhibition of Ras signalling during vulval induction is controlled by two redundant pathways, the class A and class B SynMuv genes. Further investigating this correlation, we see a silenced-transgene phenotype in non-vulval tissue in several other class B mutants, suggesting that the role of class B genes is more widespread than just in the inhibition of Ras signalling during vulval development. Our present studies are focused on understanding the connection between repetitive contexts and gene silencing and on the role of tam-1 in these processes. 1 Kelly et al Genetics 46: 227, 2 Chang et al 1997 C. elegans Meeting: 619, 3 Liu, J. CalTech Thesis (1998)

Hsieh EJ et al. (2013) J Am Soc Mass Spectrom "Effects of column and gradient lengths on peak capacity and peptide identification in nanoflow LC-MS/MS of complex proteomic samples."

MacCoss MJ, Durand S, Hsieh EJ, Valaskovic GA, Bereman MS

[J Am Soc Mass Spectrom, 2013]

Reversed-phase liquid chromatography is the most commonly used separation method for shotgun proteomics. Nanoflow chromatography has emerged as the preferred chromatography method for its increased sensitivity and separation. Despite its common use, there are a wide range of parameters and conditions used across research groups. These parameters have an effect on the quality of the chromatographic separation, which is critical to maximizing the number of peptide identifications and minimizing ion suppression. Here we examined the relationship between column lengths, gradient lengths, peptide identifications, and peptide peak capacity. We found that while longer column and gradient lengths generally increase peptide identifications, the degree of improvement is dependent on both parameters and is diminished at longer column and gradients. Peak capacity, in comparison, showed a more linear increase with column and gradient lengths. We discuss the discrepancy between these two results and some of the considerations that should be taken into account when deciding on the chromatographic conditions for a proteomics experiment.

McGhee JD (1987) Biochemistry "Purification and characterization of a carboxylesterase from the intestine of the nematode Caenorhabditis elegans."

McGhee JD

[Biochemistry, 1987]

The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].

Murakami S et al. (1999) Curr Biol "Life extension and stress resistance in Caenorhabditis elegans modulated by the tkr-1 gene."

Johnson TE, Murakami S

[Curr Biol, 1999]

In this Brief Communication, which appeared in the 14 September 1998 issue of Current Biology, the UV dose was reported erroneously. The dose reported was 20 J/m2 but the actual dose used was 0.4 J/cm2. Also, the gene formally referred to as tkr-1 has since been renamed old-1 (overexpression longevity determination).

Ramos CG et al. (2014) J Bacteriol "Retraction for Ramos et al., MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia."

Feliciano JR, da Costa PJ, Ramos CG, Grilo AM, Leitao JH

[J Bacteriol, 2014]

Volume 195, no. 16, p. 35143523, 2013. A number of problems related to images published in this paper have been brought to our attention. Figure 1D contains duplicated images in lanes S and LE, and Fig. 4D and 6B contain images previously published in articles in this journal and in Microbiology and Microbial Pathogenesis, i.e., the following: C. G. Ramos, S. A. Sousa, A. M. Grilo, J. R. Feliciano, and J. H. Leitao, J. Bacteriol. 193:15151526, 2011. doi:10.1128/JB.01374-11. S. A. Sousa, C. G. Ramos, L. M. Moreira, and J. H. Leitao, Microbiology 156:896908, 2010. doi:10.1099/mic.0.035139-0. C. G. Ramos, S. A. Sousa, A. M. Grilo, L. Eberl, and J. H. Leitao, Microb. Pathog. 48:168177, 2010. doi: 10.1016/j.micpath.2010.02.006. Therefore, we retract the paper. We deeply regret this situation and apologize for any inconvenience to the editors and readers of Journal of Bacteriology, Microbial Pathogenesis, and Microbiology.

Nillegoda NB et al. (2015) Nature "Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation."

Berynskyy M, Morimoto RI, Bukau B, Stengel F, Kirstein J, Szlachcic A, Arnsburg K, Stank A, Scior A, Nillegoda NB, Gao X, Guilbride DL, Aebersold R, Wade RC, Mayer MP

[Nature, 2015]

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.