Untranslated Segment Research Articles

Next-Generation Sequencing Identifies Extended HLA Class I and II Haplotypes Associated With Early-Onset and Late-Onset Myasthenia Gravis in Italian, Norwegian, and Swedish Populations.

Genetic susceptibility to myasthenia gravis (MG) associates with specific HLA alleles and haplotypes at the class I and II regions in various populations. Previous studies have only examined alleles at a limited number of HLA loci that defined only broad serotypes or alleles defined at the protein sequence level. Consequently, genetic variants in noncoding and untranslated HLA gene segments have not been fully explored but could also be important determinants for MG. To gain further insight into the role of HLA in MG, we applied next-generation sequencing to analyze sequence variation at eleven HLA genes in early-onset (EO) and late-onset (LO) non-thymomatous MG patients positive for the acetylcholine receptor (AChR) antibodies and ethnically matched controls from Italy, Norway, and Sweden. For all three populations, alleles and haplotype blocks present on the ancestral haplotype AH8.1 were associated with risk in AChR-EOMG patients. HLA-B*08:01:01:01 was the dominant risk allele in Italians (OR = 3.28, P = 1.83E−05), Norwegians (OR = 3.52, P = 4.41E−16), and in Swedes HLA-B*08:01 was the primary risk allele (OR = 4.24, P <2.2E-16). Protective alleles and haplotype blocks were identified on the HLA-DRB7, and HLA-DRB13.1 class II haplotypes in Italians and Norwegians, whereas in Swedes HLA-DRB7 exhibited the main protective effect. For AChR-LOMG patients, the HLA-DRB15.1 haplotype and associated alleles were significantly associated with susceptibility in all groups. The HLA-DR13–HLA-DR–HLA-DQ haplotype was associated with protection in all AChR-LOMG groups. This study has confirmed and extended previous findings that the immunogenetic predisposition profiles for EOMG and LOMG are distinct. In addition, the results are consistent with a role for non-coding HLA genetic variants in the pathogenesis of MG.

IGF2BP2/IMP2-Deficient Mice Resist Obesity through Enhanced Translation of Ucp1 mRNA and Other mRNAs Encoding Mitochondrial Proteins

Although variants in the IGF2BP2/IMP2 gene confer risk for type 2 diabetes, IMP2, an RNA binding protein, is not known to regulate metabolism. Imp2(-/-) mice gain less lean mass after weaning and have increased lifespan. Imp2(-/-) mice are highly resistant to diet-induced obesity and fatty liver and display superior glucose tolerance and insulin sensitivity, increased energy expenditure, and better defense of core temperature on cold exposure. Imp2(-/-) brown fat and Imp2(-/-) brown adipocytes differentiated in vitro contain more UCP1 polypeptide than Imp2(+/+) despite similar levels of Ucp1 mRNA; the Imp2(-/-)adipocytes also exhibit greater uncoupled oxygen consumption. IMP2 binds the mRNAs encoding Ucp1 and other mitochondrial components, and most exhibit increased translational efficiency in the absence of IMP2. In vitro IMP2 inhibits translation of mRNAs bearing the Ucp1 untranslated segments. Thus IMP2 limits longevity and regulates nutrient and energy metabolism in the mouse by controlling the translation of its client mRNAs.

The 5′ and 3′ ends of alphavirus RNAs – Non-coding is not non-functional

The non-coding regions found at the 5′ and 3′ ends of alphavirus genomes regulate viral gene expression, replication, translation and virus–host interactions, which have significant implications for viral evolution, host range, and pathogenesis. The functions of these non-coding regions are mediated by a combination of linear sequence and structural elements. The capped 5′ untranslated region (UTR) contains promoter elements, translational regulatory sequences that modulate dependence on cellular translation factors, and structures that help to avoid innate immune defenses. The polyadenylated 3′ UTR contains highly conserved sequence elements for viral replication, binding sites for cellular miRNAs that determine cell tropism, host range, and pathogenesis, and conserved binding regions for a cellular protein that influences viral RNA stability. Nonetheless, there are additional conserved elements in non-coding regions of the virus (e.g., the repeated sequence elements in the 3′ UTR) whose function remains obscure. Thus, key questions remain as to the function of these short yet influential untranslated segments of alphavirus RNAs.

Translational Repression of NhaR, a Novel Pathway for Multi-Tier Regulation of Biofilm Circuitry by CsrA

The RNA binding protein CsrA (RsmA) represses biofilm formation in several proteobacterial species. In Escherichia coli, it represses the production of the polysaccharide adhesin poly-β-1,6-N-acetyl-D-glucosamine (PGA) by binding to the pgaABCD mRNA leader, inhibiting pgaA translation, and destabilizing this transcript. In addition, CsrA represses genes responsible for the synthesis of cyclic di-GMP, an activator of PGA production. Here we determined that CsrA also represses NhaR, a LysR-type transcriptional regulator which responds to elevated [Na(+)] and alkaline pH and activates the transcription of the pgaABCD operon. Gel shift studies revealed that CsrA binds at two sites in the 5' untranslated segment of nhaR, one of which overlaps the Shine-Dalgarno sequence. An epitope-tagged NhaR protein, expressed from the nhaR chromosomal locus, and an nhaR posttranscriptional reporter fusion (PlacUV5-nhaR'-'lacZ) both showed robust repression by CsrA. Northern blotting revealed a complex transcription pattern for the nhaAR locus. Nevertheless, CsrA did not repress nhaR mRNA levels. Toeprinting assays showed that CsrA competes effectively with the ribosome for binding to the translation initiation region of nhaR. Together, these findings indicate that CsrA blocks nhaR translation. Epistasis studies with a pgaA-lacZ transcriptional fusion confirmed a model in which CsrA indirectly represses pgaABCD transcription via NhaR. We conclude that CsrA regulates the horizontally acquired pgaABCD operon and PGA biosynthesis at multiple levels. Furthermore, nhaR repression exemplifies an expanding role for CsrA as a global regulator of stress response systems.

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova & Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova & Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009). Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

Renal Medullary MicroRNAs in Dahl Salt-Sensitive Rats

MicroRNAs are endogenous repressors of gene expression. We examined microRNAs in the renal medulla of Dahl salt-sensitive rats and consomic SS-13(BN) rats. Salt-induced hypertension and renal injury in Dahl salt-sensitive rats, particularly medullary interstitial fibrosis, have been shown previously to be substantially attenuated in SS-13(BN) rats. Of 377 microRNAs examined, 5 were found to be differentially expressed between Dahl salt-sensitive rats and consomic SS-13(BN) rats receiving a high-salt diet. Real-time PCR analysis demonstrated that high-salt diets induced substantial upregulation of miR-29b in the renal medulla of SS-13(BN) rats but not in SS rats. miR-29b was predicted to regulate 20 collagen genes, matrix metalloproteinase 2 (Mmp2), integrin beta1 (Itgb1), and other genes related to the extracellular matrix. Expression of 9 collagen genes and Mmp2 was upregulated by a high-salt diet in the renal medulla of SS rats, but not in SS-13(BN) rats, an expression pattern opposite to miR-29b. Knockdown of miR-29b in the kidneys of SS-13(BN) rats resulted in upregulation of several collagen genes. miR-29b reduced expression levels of several collagen genes and Itgb1 in cultured rat renal medullary epithelial cells. Moreover, miR-29b suppressed the activity of luciferase when the reporter gene was linked to a 3'-untranslated segment of collagen genes Col1a1, Col3a1, Col4a1, Col5a1, Col5a2, Col5a3, Col7a1, Col8a1, Mmp2, or Itgb1 but not Col12a1. The result demonstrated broad effects of miR-29b on a large number of collagens and genes related to the extracellular matrix and suggested involvement of miR-29b in the protection from renal medullary injury in SS-13(BN) rats.

Cap-Independent Translation Is Required for Starvation-Induced Differentiation in Yeast

Cellular internal ribosome entry sites (IRESs) are untranslated segments of mRNA transcripts thought to initiate protein synthesis in response to environmental stresses that prevent canonical 5' cap-dependent translation. Although numerous cellular mRNAs are proposed to have IRESs, none has a demonstrated physiological function or molecular mechanism. Here we show that seven yeast genes required for invasive growth, a developmental pathway induced by nutrient limitation, contain potent IRESs that require the initiation factor eIF4G for cap-independent translation. In contrast to the RNA structure-based activity of viral IRESs, we show that an unstructured A-rich element mediates internal initiation via recruitment of the poly(A) binding protein (Pab1) to the 5' untranslated region (UTR) of invasive growth messages. A 5'UTR mutation that impairs IRES activity compromises invasive growth, which indicates that cap-independent translation is required for physiological adaptation to stress.

Association between the ACCN1 Gene and Multiple Sclerosis in Central East Sardinia

Multiple genome screens have been performed to identify regions in linkage or association with Multiple Sclerosis (MS, OMIM 126200), but little overlap has been found among them. This may be, in part, due to a low statistical power to detect small genetic effects and to genetic heterogeneity within and among the studied populations. Motivated by these considerations, we studied a very special population, namely that of Nuoro, Sardinia, Italy. This is an isolated, old, and genetically homogeneous population with high prevalence of MS. Our study sample includes both nuclear families and unrelated cases and controls. A multi-stage study design was adopted. In the first stage, microsatellites were typed in the 17q11.2 region, previously independently found to be in linkage with MS. One significant association was found at microsatellite D17S798. Next, a bioinformatic screening of the region surrounding this marker highlighted an interesting candidate MS susceptibility gene: the Amiloride-sensitive Cation Channel Neuronal 1 (ACCN1) gene. In the second stage of the study, we resequenced the exons and the 3′ untranslated (UTR) region of ACCN1, and investigated the MS association of Single Nucleotide Polymorphisms (SNPs) identified in that region. For this purpose, we developed a method of analysis where complete, phase-solved, posterior-weighted haplotype assignments are imputed for each study individual from incomplete, multi-locus, genotyping data. The imputed assignments provide an input to a number of proposed procedures for testing association at a microsatellite level or of a sequence of SNPs. These include a Mantel-Haenszel type test based on expected frequencies of pseudocase/pseudocontrol haplotypes, as well as permutation based tests, including a combination of permutation and weighted logistic regression analysis. Application of these methods allowed us to find a significant association between MS and the SNP rs28936 located in the 3′ UTR segment of ACCN1 with p = 0.0004 (p = 0.002, after adjusting for multiple testing). This result is in tune with several recent experimental findings which suggest that ACCN1 may play an important role in the pathogenesis of MS.

The untranslated regions of genes from Trypanosoma cruzi: perspectives for functional characterization of strains and isolates

The sequencing of Trypanosoma cruzi genome has been completed and a great deal of information is now available. However, the organization of protozoa genomes is somewhat elusive and much effort must be applied to reveal all the information coded in the nucleotide sequences. Among the DNA segments that needs further investigation are the untranslated regions of genes. Many of the T. cruzi genes that were revealed by the genome sequencing lack information about the untranslated regions. In this paper, some features of these untranslated segments as well as their applications in T. cruzi populations are discussed.

Modulation of the two-pore domain acid-sensitive K+ channel TASK-2 (KCNK5) by changes in cell volume.

The molecular identity of K(+) channels involved in Ehrlich cell volume regulation is unknown. A background K(+) conductance is activated by cell swelling and is also modulated by extracellular pH. These characteristics are most similar to those of newly emerging TASK (TWIK-related acid-sensitive K(+) channels)-type of two pore-domain K(+) channels. mTASK-2, but not TASK-1 or -3, is present in Ehrlich cells and mouse kidney tissue from where the full coding sequences were obtained. Heterologous expression of mTASK-2 cDNA in HEK-293 cells generated K(+) currents in the absence intracellular Ca(2+). Exposure to hypotonicity enhanced mTASK-2 currents and osmotic cell shrinkage led to inhibition. This occurred without altering voltage dependence and with only slight decrease in pK(a) in hypotonicity but no change in hypertonicity. Replacement with other cations yields a permselectivity sequence for mTASK-2 of K(+) > Rb(+) Cs(+) > NH(4)(+) > Na(+) congruent with Li(+), similar to that for the native conductance (I(K, vol)). Clofilium, a quaternary ammonium blocker of I(K, vol), blocked the mTASK-2-mediated K(+) current with an IC(50) of 25 microm. The presence of mTASK-2 in Ehrlich cells, its functional similarities with I(K, vol), and its modulation by changes in cell volume suggest that this two-pore domain K(+) channel participates in the regulatory volume decrease phenomenon.

Nucleotide-sequence-specific and non-specific interactions of T4 DNA polymerase with its own mRNA.

The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding protein that represses translation of its own mRNA. Previous studies implicated two segments of the untranslated 5'-leader of the mRNA in repressor binding, an RNA hairpin structure and the adjacent RNA to the 3' side, which contains the Shine-Dalgarno sequence. Here, we show by in vitro gp43-RNA binding assays that both translated and untranslated segments of the mRNA contribute to the high affinity of gp43 to its mRNA target (translational operator), but that a Shine-Dalgarno sequence is not required for specificity. Nucleotide sequence specificity appears to reside solely in the operator's hairpin structure, which lies outside the putative ribosome-binding site of the mRNA. In the operator region external to the hairpin, RNA length rather than sequence is the important determinant of the high binding affinity to the protein. Two aspects of the RNA hairpin determine specificity, restricted arrangement of purine relative to pyrimidine residues and an invariant 5'-AC-3' in the unpaired (loop) segment of the RNA structure. We propose a generalized structure for the hairpin that encompasses these features and discuss possible relationships between RNA binding determinants of gp43 and DNA binding by this replication enzyme.

Genomic organization of the 3' region of the human thyroglobulin gene.

The genomic organization of the 3' end of the human Thyroglobulin (Tg) gene has not previously been characterized. We isolated and characterized seventeen lambda phage clones from a human genomic library that included nucleotides 6263 to 8410 of the Tg mRNA, encompassing the last thirteen 3' exons of the Tg gene. The region contained exons ranging in size from 94 to 222 nucleotides, split by introns of 1 to 64 kb. We estimate a total of 48 exons in the Tg gene. All the intron-exon boundaries were sequenced. We found that the splicing sequences diverged considerably from the 3' and 5' consensus. However, the GT-AG rule was perfectly respected in all the exons. A total of 5788 intronic bases and most of the sequences contained in the 13 exons were analyzed (1846 bases). One sequence variation, TT to CC at positions 8377-8378, was found in the 3' untranslated segment. The three tyrosine residues involved in thyroid hormones synthesis (amino acids 2554, 2568, and 2747) at the carbosyl termini of Tg, are encoded by exons 44, 45, and 48. The knowledge of the precise organization of the Tg gene should help to direct studies of Tg gene mutations in families in which a defect in the synthesis of Tg occurs.

Cloning and Expression of Canine Glycoprotein Ibα

The interaction of the glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (vWF) is critical in initiation of haemostasis and thrombosis through platelet adhesion to damaged endothelium. The binding site for vWF resides within the GPIbalpha subunit of the complex. To further define the physiological function of platelet GPIbalpha we cloned and expressed the canine GPIbalpha cDNA. A canine platelet cDNA library was constructed and screened with a randomly primed 32P-labeled 1041-base-pair restriction fragment of the human GPIbalpha cDNA. Analysis of 23 clones demonstrated that the canine GPIbalpha cDNA is 2530 nucleotides in length and includes a short 5' untranslated segment of 42 nucleotides followed by a signal peptide of 16 amino acids, a mature peptide of 645 amino acids and a 3' noncoding region of 455 nucleotides. A single intron of 142 nucleotides, 6 nucleotides upstream from the ATG translation initiation codon was identified in the canine gene in a similar location to that present in the human gene. Chinese hamster ovary cells that stably express human GPIbbeta and GPIX were transfected with the canine GPIbalpha cDNA. Canine GPIbalpha was expressed on the surface of these cells and bound vWF in the presence of botrocetin. The binding of vWF was inhibited by an anti-vWF human monoclonal antibody known to inhibit vWF binding to GPIbalpha. The results of this investigation will allow the development of reagents to study the physiological function of GPIbalpha in an animal model.

Tsetse thrombin inhibitor: bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans.

The tsetse thrombin inhibitor, a potent and specific low molecular mass (3,530 Da) anticoagulant peptide, was purified previously from salivary gland extracts of Glossina morsitans morsitans (Diptera: Glossinidae). A 303-bp coding sequence corresponding to the inhibitor has now been isolated from a tsetse salivary gland cDNA library by using degenerate oligonucleotide probes. The full-length cDNA contains a 26-bp untranslated segment at its 5' end, followed by a 63-bp sequence corresponding to a putative secretory signal peptide. A 96-bp segment codes for the mature tsetse thrombin inhibitor, whose predicted molecular weight matches that of the purified native protein. Based on its lack of homology to any previously described family of molecules, the tsetse thrombin inhibitor appears to represent a unique class of naturally occurring protease inhibitors. Recombinant tsetse thrombin inhibitor expressed in Escherichia coli and the chemically synthesized peptide are both substantially less active than the purified native protein, suggesting that posttranslational modification(s) may be necessary for optimal inhibitory activity. The tsetse thrombin inhibitor gene, which is present as a single copy in the tsetse genome, is expressed at high levels in salivary glands and midguts of adult tsetse flies, suggesting a possible role for the anticoagulant in both feeding and processing of the bloodmeal.

Molecular, enzymatic, and regulatory characterization of rat kidney cytochromes P450 4A2 and 4A3.

The cDNAs encoding cytochromes CYP 4A2 and 4A3 were cloned by RT-PCR amplification of male rat kidney and liver RNAs, respectively. Sequence analysis demonstrated that these cDNAs were nearly identical to the published sequences for CYPs 4A2 and 4A3. CYP 4A2 and 4A3 share extensive sequence homology that extends into their 3'- and 5'-untranslated segments ( approximately 97% overall nucleotide identity). Analysis of cDNA and genomic DNA sequences shows that a sequence of 123 bp, recognized as an intron during the processing of CYP 4A2 transcripts, is conserved in the 4A3 mRNAs and that these otherwise highly homologous genes show different exon-intron distributions. The CYP 4A2 and 4A3 cDNAs were expressed in a baculovirus-insect cell expression system. Purified recombinant CYP 4A2 oxidized arachidonic acid to a mixture of 19- and 20-hydroxyeicosatetraenoic acids (20 and 80% of the total products, respectively). Reaction rates were maximal when CYP 4A2 was reconstituted in the presence of an equimolar concentration of cytochrome b5 and a 10-fold molar excess of NADPH-cytochrome P450 reductase. Studies using microsomal fractions isolated from noninfected insect cells and from cells infected with CYP 4A3 recombinant baculoviruses showed (a) the presence of an endogenous lauric acid omega-hydroxylase and arachidonic acid epoxygenase in the noninfected cells, (b) the CYP 4A3-dependent oxidation of lauric acid to 11- and 12-hydroxylaurate (24 and 76% of the total products, respectively), and (c) the lack of arachidonic acid metabolism by microsomal recombinant CYP 4A3. Nucleic acid hybridization and immunoelectrophoresis studies demonstrated that (a) CYP 4A2 transcripts are abundantly expressed in the female kidney and that CYP 4A3 is expressed in female but not in male liver, (b) anti-CYP 4A2 immunoreactive material was detected only in the male kidney, (c) male and female livers or kidneys support only low levels of CYP 4A3 translation, and (d) excess dietary salt does not alter the kidney levels of mRNA transcripts encoding CYP 4A1, 4A2, or 4A3 or change the levels of microsomal anti-4A1 or -4A2 immunoreactive proteins. Finally, no significant differences were observed between Dahl salt resistant or Dahl salt sensitive rats in the levels and/or salt regulation of mRNA transcripts enecoding CYP 4A1, 4A2, or 4A3 or the in levels of the corresponding proteins.

Exon/intron structure of the human transferrin receptor gene

A PCR-based intron jumping strategy has been utilized to investigate the exon/intron structure of the human transferrin receptor gene and determine the sequences of exon/intron junctions. There are 18 exons and introns 5′ to a large exon encoding the last translated segment and a sizable 3′ untranslated segment. All of the translated segments are encoded by exons 2–19. The tight turn motif, which is critical to the process of endocytosis, is encoded by exon 3. Based on recent studies of human/chicken receptor chimeras, it appears that the residues most likely to be involved in transferrin binding are encoded by exons 17–19. Exon 12 exhibits the greatest degree of homology with the gene for the prostate specific membrane antigen. A polymorphism has been tentatively identified at nucleotide position 519 in exon 4; the presence of either adenine or guanine should result in either serine or glycine, respectively, at position 142 of the amino acid sequence. This analysis of genomic structure will permit further detailed studies of the regulation, expression and evolution of the human transferrin receptor gene.

Molecular characterization of coding and untranslated regions of rat cortex lithium-sensitive myo-inositol monophosphatase cDNA

Lithium sensitive myo-inositol monophosphatase (IMPase) is a pivotal enzyme which controls the levels of brain inositol within the inositol-based signaling system. Its capacity to release free myo-inositol from inositol monophosphates generated from receptor-linked and de novo pathways is crucial to the maintenance of appropriate amounts of intracellular myo-inositol, which is essential for both inositol-based cell signaling and cell volume control. We present here the full length cDNA encompassing the coding and untranslated regions (5′- and 3′-UTRs) of rat brain IMPase. This cDNA was derived from rat cortex mRNA by the RT-PCR technique. Analysis of this cDNA revealed several interesting features which include a short 5′-untranslated region (5′-UTR) of 68 nucleotides followed by coding region of approximately 0.8 kb and a long 3′-untranslated region (3′-UTR) of 1.2 kb. Both 5′-rapid amplification of cDNA ends (5′-RACE) and 3′-RACE techniques were carried out to isolate both UTRs and double stranded sequencing was carried out to its entirety (∼2.1 kb) by `gene walking' using several oligonucleotide primers. All nucleotides were sequenced unambiguously using the sense and antisense strands of DNA. PCR analysis for the coding region and the deduced amino acid sequence demonstrated a DNA fragment of 831 bp and 277 amino acids, respectively, which are strikingly similar to human hippocampal IMPase. The 5′-UTR demonstrated distinct CpG doublets, characteristic of `housekeeping' genes. The sequence around the initiator methionine, AAGATGG, conforms well to the Kozak consensus sequence for mammalian protein biosynthesis and the 3′-UTR demonstrated three canonical ( AATAAT , AATTAA , AATACA ) and one unusual poly-adenylation signals ( ATTAAA ) followed by a 31 base poly(A) tail. The presence of a CCTGTG in the 3′-UTR (putative carbohydrate response element) links IMPase mRNA to brain carbohydrate metabolic pathways. Computer analyses demonstrated several unique features of this mRNA, including the potential formation of hairpin loops which might be important in its intracellular regulation and turn-over. In summary, this lithium-sensitive brain IMPase mRNA has the following characteristics: a 5′-CpG-rich short untranslated segment, a highly conserved coding region, and a long 3′-untranslated region with several polyadenylation signals. © 1997 Elsevier Science B.V. All rights reserved.

Identification and Characterization of Galectin-9, a Novel β-Galactoside-binding Mammalian Lectin

A 36-kDa beta-galactoside mammalian lectin protein, designated as galectin-9, was isolated from mouse embryonic kidney by using a degenerate primer polymerase chain reaction and cloning strategy. Its deduced amino acid sequence had the characteristic conserved sequence motif of galectins. Endogenous galectin-9, extracted from liver and thymus, as well as recombinant galectin-9 exhibited specific binding activity for the lactosyl group. It had two distinct N- and C-terminal carbohydrate-binding domains connected by a link peptide, with no homology to any other protein. Galectin-9 had an alternate splicing isoform, exclusively expressed in the small intestine with a 31-amino acid insertion between the N-terminal domain and link peptide. Sequence homology analysis revealed that the C-terminal carbohydrate-binding domain of mouse galectin-9 had extensive similarity to that of monomeric rat galectin-5. The presence of galectin-5 in the mouse could not be demonstrated by polymerase chain reaction or by Northern or Southern blot genomic DNA analyses. Sequence comparison of rat galectin-5 and rat galectin-9 cDNA did not reveal identical nucleotide sequences in the overlapping C-terminal carbohydrate-binding domain, indicating that galectin-9 is not an alternative splicing isoform of galectin-5. However, galectin-9 had a sequence identical with that of its intestinal isoform in the overlapping regions in both species. Southern blot genomic DNA analyses, using the galectin-9 specific probe derived from the N-terminal carbohydrate-binding domain, indicated the presence of a novel gene encoding galectin-9 in both mice and rats. In contrast to galectin-5, which is mainly expressed in erythrocytes, galectin-9 was found to be widely distributed, i.e. in liver, small intestine, thymus > kidney, spleen, lung, cardiac and skeletal muscle > reticulocyte, brain. Collectively, these data indicate that galectin-9 is a new member of the galectin gene family and has a unique intestinal isoform.

Mosquito dopa decarboxylase cDNA characterization and blood‐meal‐induced ovarian expression

Dopa decarboxylase (DDC) functions in insect catecholamine biochemistry to produce materials essential for cross-linking reactions that result in tanning and/or melanization, include tanning of the mosquito egg chorion and encapsulation of parasites. We have cloned Ddc from the mosquito, Aedes aegypti, and studied its expression in response to blood-feeding, which initiates events necessary for egg maturation in mosquitoes. The Ae. aegypti Ddc cDNA was isolated via heterologous screening using a clone from Drosophila melanogaster. A resulting 1.87 kilobase (kb) clone was sequenced to reveal an open reading frame of 1464 bp, as well as 5'- and 3'-untranslated segments. The inferred amino acid sequence of this clone shares 81% identity with the published Drosophila Ddc cDNA, including complete identity with twenty-four contiguous amino acids encompassing the pyridoxal-5-phosphate cofactor binding domain. Analysis of an F2 intercross population derived from a parental cross between two Ae. aegypti strains (Hamburg and Moyo-In-Dry) allowed us to map Ddc to a locus on linkage group 2. Expression studies demonstrated the presence of a 2.1 kb message, the majority of which occurs in the ovaries where Ddc-specific mRNA is up-regulated in response to ingestion of a blood meal. The potential for egg-tanning in anautogenous mosquitoes as a model for understanding specific genetic events in the regulation of catecholamine metabolism is addressed.