Carbon nanotube fibers are compatible with Mammalian cells and neurons.

We demonstrate the biocompatibility of carbon nanotube fibers (CNFs) fabricated from single-wall carbon nanotubes. Produced by a particle-coagulation spinning process, CNFs are "hair-like" conductive microwires, which uniquely combine properties of porous nanostructured scaffolds, high-area electrodes, and permeable microfluidic conduits. We report that CNFs are nontoxic and support the attachment, spreading, and growth of mammalian cells and the extension of processes from neurons in vitro. Our findings suggest that CNF may be employed for an electrical interfacing of nerve cells and external devices.

Structural and functional analysis of the MAL1 locus of Saccharomyces cerevisiae.

We describe the isolation of a 22.6-kilobase fragment of DNA containing the MAL1 locus of Saccharomyces cerevisiae. Our results demonstrate that the MAL1 locus, like the MAL6 locus, is a complex locus containing three genes. These genes were organized similarly to their MAL6 counterparts. We refer to them as MAL11, MAL12, and MAL13 and show that they are functionally homologous to the MAL61 (encoding maltose permease), MAL62 (encoding maltase), and MAL63 (encoding the positive regulator) genes of the MAL6 locus. Transcription from each of the three genes was analyzed in a strain carrying the undisrupted MAL1 locus and in strains carrying single disruptions in each of the MAL1 genes. The MAL1 and MAL1 loci were found to be highly sequence homologous and conserved throughout the region containing these three genes. The strain used to isolate the MAL1 locus also carried the tightly linked SUC1 gene. The SUC1 gene was found to be located on the same 22.6-kilobase fragment containing the MAL1 locus and 5 kilobases from the 3' end of the MAL12 gene. The meaning of these results with regard to the mechanism of regulation of maltose fermentation is discussed.

Expression of the murine alpha B-crystallin gene is not restricted to the lens.

The murine alpha B-crystallin gene was cloned and its expression was examined. In the mouse, significant levels of alpha B-crystallin RNA were detected not only in lens but also in heart, skeletal muscle, kidney, and lung; low and trace levels were detected in brain and spleen, respectively. The RNA species in lung, brain, and spleen was 400 to 500 bases larger than that in the other tissues. Transcription in lens, heart, skeletal muscle, kidney, and brain initiated at the same position. A mouse alpha B-crystallin mini-gene was constructed and was introduced into the germ line of mice, and its expression was demonstrated to parallel that of the endogenous gene. Transgene RNA was always detected in lens, heart, and skeletal muscle, while expression in kidney and lung was variable; it remains uncertain whether there is transgene expression in brain and spleen. These results demonstrate that regulatory sequences controlling expression of the alpha B-crystallin gene lie between sequences 666 base pairs upstream of the transcription initiation site and 2.4 kilobase pairs downstream of the poly(A) addition site and are not located within the introns. Transfection studies with a series of alpha B-crystallin mini-gene deletion mutants revealed that sequences between positions -222 and -167 were required for efficient expression in primary embryonic chick lens cells; sequences downstream of the poly(A) addition signal were dispensable for expression in this in vitro system.

Expression of the murine alpha B-crystallin gene in lens and skeletal muscle: identification of a muscle-preferred enhancer.

The alpha B-crystallin gene is expressed at high levels in lens and at lower levels in some other tissues, notably skeletal and cardiac muscle, kidney, lung, and brain. A promoter fragment of the murine alpha B-crystallin gene extending from positions -661 to +44 and linked to the bacterial chloramphenicol acetyltransferase (CAT) gene showed preferential expression in lens and skeletal muscle in transgenic mice. Transfection experiments revealed that a region between positions -426 and -257 is absolutely required for expression in C2C12 and G8 myotubes, while sequences downstream from position -115 appear to be determinants for lens expression. In association with a heterologous promoter, a -427 to -259 fragment functions as a strong enhancer in C2C12 myotubes and less efficiently in myoblasts and lens. Gel shift and methylation interference studies demonstrated that nuclear proteins from C2C12 myoblasts and myotubes specifically bind to the enhancer.

Identification of a second trans-acting gene controlling maltose fermentation in Saccharomyces carlsbergensis.

Maltose fermentation in Saccharomyces spp. requires the presence of a dominant MAL locus. The MAL6 locus has been cloned and shown to encode the structural genes for maltose permease (MAL61), maltase (MAL62), and a positively acting regulatory gene (MAL63). Induction of the MAL61 and MAL62 gene products requires the presence of maltose and the MAL63 gene. Mutations within the MAL63 gene produce nonfermenting strains unable to induce the two structural gene products. Reversion of these mal63 nonfermenters to maltose fermenters nearly always leads to the constitutive expression of maltase and maltose permease, and constitutivity is always linked to MAL6. We demonstrated that for one such revertant, strain C2, constitutivity did not require the MAL63 gene, since deletion disruption of this gene did not affect the constitutive expression of the structural genes. In addition, constitutivity was trans acting. Deletion disruption of the MAL6-linked structural genes for maltase and maltose permease in this strain did not affect the constitutive expression of a second, unlinked maltase structural gene. We isolated new maltose-fermenting revertants of a nonfermenting strain which carried a deletion disruption of the MAL63 gene. All 16 revertants isolated expressed maltase constitutively. In one revertant studied in detail, strain R10, constitutive expression was demonstrated to be linked to MAL6, semidominant, trans acting, and residing outside the MAL63-MAL61-MAL62 genes. From these studies we propose the existence of a second trans-acting regulatory gene at the MAL6 locus. We call this new gene MAL64. We mapped the MAL64 gene 2.3 centimorgans to the left of MAL63. The role of the MAL64 gene product in maltose fermentation is discussed.

Constitutive expression of the maltose fermentative enzymes in Saccharomyces carlsbergensis is dependent upon the mutational activation of a nonessential homolog of MAL63.

Maltose fermentation in Saccharomyces carlsbergensis is dependent upon the MAL6 locus. This complex locus is composed of the MAL61 and MAL62 genes, which encode maltose permease and maltase, respectively, and a third gene, MAL63, which codes for a trans-acting positive regulatory product. In wild-type strains, expression of the MAL61 and MAL62 mRNAs and proteins is induced by maltose and induction is dependent upon the MAL63 gene. Mutants constitutively expressing the MAL61 and MAL62 gene products have been isolated in mal63 backgrounds, and the mutations which have been analyzed map to a fourth MAL6-linked gene, MAL64. Cloning and characterization of this new gene are described in this report. The results revealed that the MAL64-C alleles present in constitutive strains encode a trans-acting positive function required for constitutive expression of the MAL61 and MAL62 gene products. In inducible strains, the MAL64 gene is dispensable, as deletion of the gene had no effect on maltose fermentation or maltose-regulated induction. MAL64 encoded transcripts of 2.0 and 1.4 kilobase pairs. While both MAL64 mRNAs were constitutively expressed in constitutive strains, they were maltose inducible in wild-type strains and induction was dependent upon the MAL63 gene. The MAL63 and MAL64 genes are at least partially structurally homologous, suggesting that they control MAL61 and MAL62 transcript accumulation by similar mechanisms.

Sry is a transcriptional activator.

The SRY gene functions as a genetic switch in gonadal ridge initiating testis determination. The mouse Sry and human SRY open reading frames (ORFs) share a conserved DNA-binding domain (the HMG-box) yet exhibit no additional homology outside this region. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and mouse SRY ORFs contain a nuclear localization signal. The mouse Sry HMG-box domain selectively binds the sequence NACAAT in vitro when challenged with a random pool of oligonucleotides and binds AACAAT with the highest affinity. When put under the control of a heterologous promotor, the mouse Sry gene activated transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was likewise observed for a GAL4-responsive reporter gene, when the mouse Sry gene was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a glutamine/histidine-rich domain. In addition, LexA-mouse Sry fusion genes activated a LexA-responsive reporter gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and the mouse SRY ORFs encode nuclear, DNA-binding proteins and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.

Functional comparison of the Mus musculus molossinus and Mus musculus domesticus Sry genes.

The Sry gene functions as a genetic switch initiating testicular development of the indifferent mammalian gonad. The Mus musculus molossinus Sry open reading frame (ORF) encodes a 395-amino acid transcription factor (mSry) that specifically binds and bends DNA through its N-terminal HMG domain and activates transcription through its long C-terminal (residues 144-366) glutamine/histidine-rich activation domain. The M. m. domesticus Sry ORF encodes a highly homologous, truncated protein (dSry) of approximately 230 amino acids, and the molecular basis for truncation is a point mutation that creates an amber stop codon within the activation domain. The mSry protein activates transcription of a Sry-responsive reporter gene in HeLa cells, but dSry does not. Gene swapping and in vitro DNA binding experiments revealed that lack of transcriptional activation by dSry was not the result of polymorphisms within the first 137 amino acids of the protein. Direct analysis of the C-terminal glutamine/histidine-rich domain revealed that dSry lacked a functional transcriptional activation domain. Fusion of the GAL4 DNA-binding domain to the C-terminal deletion mutants of the GAL4-mSry chimeric protein indicated that residues 263-345 of the glutamine/histidine-rich domain were necessary for high level transactivation. Furthermore, readthrough of the premature amber stop codon by transfer RNA suppression resulted in a strong GAL4-dSry transactivator. This demonstrated that the premature stop codon is the only polymorphism responsible for the inability of the dSry glutamine/histidine-rich region to transactivate.

Inverted repeats are necessary for circularization of the mouse testis Sry transcript.

Circular non-polyadenylated RNA molecules have been identified as stable transcription products of the human ETS-1 and mouse Sry genes. RNA circularization has been proposed to require two steps. The first step utilizes intramolecular base pairing to produce a transient stem-loop structure. The second step involves splicing a downstream donor splice site (DSS) to a now closely appositioned upstream acceptor splice site (ASS) within the loop. We demonstrate that the presence of long inverted repeats (IR) flanking the mouse Sry gene leads to the formation of the Sry circular transcript in cultured cells. Circularization requires the presence of both IR. As few as 400 complementary nt are necessary for this process. The presence of the IR does not significantly stimulate intermolecular annealing and trans-splicing in vivo.

High-level inducible expression of visual pigments in transfected cells.

A method for high-level expression of a functionally active, recombinant human red cone opsin was developed by adding the coding sequence for the C-terminal epitope of bovine rhodopsin onto the C terminus of the cone opsin and cloning the resulting construct into the vector pMEP4 beta. The recombinant pMEP4 beta vector was transfected stably into 293-EBNA cells, and expression of the cone opsin was induced by the addition of CdCl2 into the medium. The recombinant cone opsin was reconstituted with 11-cis retinal and purified by immunoaffinity chromatography. Spectral analysis prior to and following photobleaching confirmed its identity as a red cone opsin. The protein was targeted to the cell membrane and activated bovine transducin.

Synopsis of preterm birth genetic association studies: the preterm birth genetics knowledge base (PTBGene).

AIM: Our goal wasto produce a field synopsis of genetic associations with preterm birth and to set up a publicly available online database summarizing the data. METHODS: We performed a systematic review and meta-analyses to identify genetic associations with preterm birth. We have set up a publicly available online database of genetic association data on preterm birth called PTBGene (http://ric.einstein.yu.edu/ptbgene/index.html) and report on a structured synopsis thereof as of December 1, 2008. RESULTS: Data on 189 polymorphisms in 84 genes have been included and 36 meta-analyses have been performed. Five gene variants (4 in maternal DNA, one in newborn DNA) have shown nominally significant associations, but all have weak epidemiological credibility. CONCLUSION: After publishing this field synopsis, the PTBGene database will be regularly updated to keep track of the evolving evidence base of genetic factors in preterm birth with the goal of promoting knowledge sharing and multicenter collaboration among preterm birth research groups.

Structure and alternate tissue-preferred transcription initiation of the mouse alpha B-crystallin/small heat shock protein gene.

We have determined the complete nucleotide sequence (-865 to +3515) of the murine alpha B-crystallin/small heat shock protein gene, a major soluble protein of the vertebrate eye lens. Its 3 exon/2 intron structure is identical to that of the rat, hamster and human gene, with the exons being much more conserved than the introns. Previous reports indicated that there are two sizes of alpha B-crystallin mRNA; a larger alpha B-crystallin mRNA predominates in the lung and brain and is also found in low levels in most other tissues (except in lens and liver), while a smaller alpha B-crystallin mRNA exists at a high level in the lens and in variable amounts elsewhere. Sequence analysis suggests that secondary structure in the 5' untranslated sequence of the longer mRNA has led to difficulty in mapping the transcription initiation site of the longer transcript. Here we provide evidence by primer extension, S1 nuclease protection, and PCR (polymerase chain reaction) experiments for a transcription initiation site in the murine lung and brain at position -474. We also detected the utilization of the -474 initiation site in lens and of the +1 site in lung and brain, indicating that the tissue preference for these sites is not absolute. In vitro transcription experiments revealed that cell-free HeLa nuclear extracts specifically initiate transcription at the -474 and +1 sites. alpha B-crystallin was immunocytochemically localized to the bronchioles of the lung. Thus, regulation of alpha B-crystallin/small heat shock protein expression involves the utilization of tissue-preferred transcription initiation sites.

Human alpha B-crystallin gene and preferential promoter function in lens.

alpha B-Crystallin, first identified as a structural component of the vertebrate eye lens, is expressed at high levels in lens and at lower levels in a number of other tissues, most notably cardiac and skeletal muscle, kidney, and brain. We have cloned and sequenced the human alpha B-crystallin gene and show that it is structurally similar to its hamster homolog. We have also identified its transcription initiation site in human lens RNA. Functional analysis of a promoter fragment extending from -537 to +21 (relative to the transcription initiation site) and fused to the bacterial chloramphenicol acetyltransferase gene suggests that this fragment contains regulatory elements that function preferentially, but not exclusively, in lens. In contrast, this fragment is apparently insufficient to promote transcription in glial cells, as this construct functioned poorly in a glioblastoma-astrocytoma cell line (U-373MG) that synthesizes high levels of the endogenous alpha B-crystallin gene product.

Identification of the structural gene encoding maltase within the MAL6 locus of Saccharomyces carlsbergensis.

Saccharomyces yeast strains able to ferment maltose carry at least one member of a family of MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. The MAL6 locus has been cloned and shown to be a cluster of at least three transcribed regions, all of which are required for maltose fermentation. Transcription at two of these genes, MAL61 and MAL62, is both induced by maltose and repressed by glucose. The third gene, MAL63, appears to encode a regulatory product controlling maltose fermentation. In this report, we demonstrate that the MAL62 gene is the structural gene coding for the enzyme maltase. Strain 332-5A is a maltose fermenter of the genotype MAL6 mal1(0). Integrative disruption of the MAL62 gene of the MAL6 locus produces a strain which is still capable of fermenting maltose, but which synthesizes a more heat-labile form of maltase than the undisrupted strain. Synthesis of this more heat-labile maltase was shown to be linked to the mal1(0) locus present in the strain. Integrative disruption of both the MAL62 gene and the MAL62-homologous sequence present at the mal1(0) locus produces a nonfermenter which is unable to synthesize maltase. These results identify MAL62 as the maltase structural gene.

Identification and characterization of the maltose permease in genetically defined Saccharomyces strain.

Saccharomyces yeasts ferment several alpha-glucosides including maltose, maltotriose, turanose, alpha-methylglucoside, and melezitose. In the utilization of these sugars transport is the rate-limiting step. Several groups of investigators have described the characteristics of the maltose permease (D. E. Kroon and V. V. Koningsberger, Biochim. Biophys. Acta 204:590-609, 1970; R. Serrano, Eur. J. Biochem. 80:97-102, 1977). However, Saccharomyces contains multiple alpha-glucoside transport systems, and these studies have never been performed on a genetically defined strain shown to have only a single permease gene. In this study we isolated maltose-negative mutants in a MAL6 strain and, using a high-resolution mapping technique, we showed that one class of these mutants, the group A mutants, mapped to the MAL61 gene (a member of the MAL6 gene complex). An insertion into the N-terminal-coding region of MAL61 resulted in the constitutive production of MAL61 mRNA and rendered the maltose permease similarly constitutive. Transformation by high-copy-number plasmids containing the MAL61 gene also led to an increase in the maltose permease. A deletion-disruption of MAL61 completely abolished maltose transport activity. Taken together, these results prove that this strain has only a single maltose permease and that this permease is the product of the MAL61 gene. This permease is able to transport maltose and turanose but cannot transport maltotriose, alpha-methylglucoside, or melezitose. The construction of strains with only a single permease will allow us to identify other maltose-inducible transport systems by simple genetic tests and should lead to the identification and characterization of the multiple genes and gene products involved in alpha-glucoside transport in Saccharomyces yeasts.

MAL63 codes for a positive regulator of maltose fermentation in Saccharomyces cerevisiae.

Genetic analysis of the MAL6 locus has previously yielded mal6 mutants which fall into a single complementation group and which are noninducible for maltase and maltose permease. However, the strains used in these studies contained additional partially functional copies of MAL1 (referred to as MAL1g) and MAL3 (referred to as MAL3g). Using a strain lacking MALg genes, we have isolated two classes of mutants and these classes correspond to mutations in MAL63 and MAL61, two genes of the MAL6 complex. Disruptions of MAL63 are noninducible for maltase and maltose permease and for their corresponding mRNAs. The mal6 mutants are shown to map to MAL63. Inducer exclusion as a cause of the noninducible phenotype of the mal63 mutations has been eliminated by constructing a mal63 mutant in a strain constitutive for maltose permease; the strain remains noninducible. These results rigorously demonstrate that MAL63 is a regulatory gene which plays a positive role in the regulation of maltose fermentation.

Assignment of the alpha B-crystallin gene to human chromosome 11.

Using a human alpha B-crystallin genomic probe and human-mouse somatic cell hybrids, the human alpha B-gene was assigned to chromosome 11 and further corroborated by in situ hybridization to normal metaphase chromosomes. This assignment confirmed and regionally mapped the locus to q22.3-23.1.

Lens protein gene expression: alpha-crystallins and MIP.

The crystallin genes encode the major soluble proteins of the lens. Some of the crystallin genes are expressed exclusively in the lens while others are also expressed in different tissues. The two alpha-crystallin genes, alpha A and alpha B, differ in their tissue specificity. Transcription of the alpha A-crystallin gene occurs only in the lens, while the alpha B-crystallin gene is also expressed in other tissues, including heart, skeletal muscle, kidney, lung and brain. MIP (also called MP26), the major intrinsic protein of the lens fiber membranes, is also expressed exclusively in the lens. Correct expression of both alpha-crystallin and MIP are required for normal lens function. Here we review our studies on the molecular basis of expression of the alpha-crystallin and MIP genes in the lens. The 5' flanking sequences containing the initiation site of transcription of the alpha A-crystallin, alpha B-crystallin and MIP genes were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene, and the expression of this reporter gene was studied in transient assays and transgenic mice. DNA sequences flanking the 5' end of the alpha A-crystallin gene contain regulatory elements responsible for the lens-specific expression and developmental regulation of the CAT gene in transgenic mice. Interestingly, although some of the murine alpha A-crystallin regulatory sequences are conserved in the human and chicken genes, different functional regulatory elements appear to control the expression of the murine and chicken alpha A-crystallin genes. The 5' flanking sequence of the alpha B-crystallin gene preferentially directs expression of the CAT gene to the lens and to skeletal muscle. Different regulatory elements of the alpha B-crystallin gene appear to be responsible for its transcription in various tissues. The 5' flanking sequence of the MIP gene also contains regulatory elements that direct expression of the CAT gene to lens cells; these sequences are not functional in transfected non-lens cells and are different from the cis regulatory elements controlling alpha-crystallin gene expression. The multiplicity of cis-regulatory elements controlling the transcription of these three genes indicates the complexity of the mechanisms that regulate gene expression in the lens.

MAL6 of Saccharomyces: a complex genetic locus containing three genes required for maltose fermentation.

The MAL6 locus is one of five closely related unlinked loci, any one of which is sufficient for fermentation of maltose in Saccharomyces. Previous genetic analysis indicated that this locus is defined by two complementation groups, MALp and MALg. MALp reportedly is a regulatory gene required for inducible synthesis of the two enzymatic functions needed for fermentation: maltose permease and maltase. We have investigated the physical and genetic structure of the MAL6 locus, which has been isolated on a recombinant DNA plasmid. One subclone of the region, pDF-1, was found to encode a single transcribed region and to contain the MALp gene. A second subclone, p1, was shown to contain the MALg function but surprisingly had not one but two maltose-inducible transcripts. Subclones having only one of these transcribed regions lacked MALg activity. The three transcribed regions have been named MAL61 and MAL62, which correspond to MALg, and MAL63, which corresponds to MALp. This clustered arrangement of a regulatory gene adjacent to the sequences it controls has not previously been described in eukaryotes and is reminiscent of bacterial operons except that the messenger RNA molecules are not polycistronic.