OAKTrust :: Browsing by Author "Summers, Max D."

Browsing by Author "Summers, Max D."

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Baculovirus dual promoter expression vector
(United States. Patent and Trademark Office, 1992-12-08) Summers, Max D.; Oker-Blom, Christian E. G.; The Texas A & M University System
This invention relates to specifically designing and genetically engineering recombinant baculovirus for producing, in a compatible insect system, a desired protein, virus, protein hybrid, or virus hybrid. In particular aspects, this invention relates to the use of different baculovirus promoters for the ultimate purpose of constructing a recombinant baculovirus designed for the investigator's specific need. For example, the recombinant baculovirus of this invention can be designed to produce a viral pesticide.This invention also describes the construction of a genetically engineered virus or virus hybrid (e.g. animal or human pathogen) which is not capable of replicating itself but is essentially identical to the authentic pathogen in terms of structure and antigenicity. This baculovirus is constructed such that the non-structural viral genes are truncated, mutated or both and are located 3' and directly under the control of an early baculovirus gene promoter and the structural viral genes are located 3' and directly under the control of a late baculovirus gene promoter. This genetically engineered baculovirus is therefore capable of temporal regulation and successive synthesis of non-structural and structural proteins. The truncated or mutated non-structural viral genes creates the non-replicative aspect of this invention. Since the genetically produced virus or virus hybrid is essentially identical to the authentic pathogen, the product is thereby highly antigenic and potent in terms of efficacy and efficiency. This invention enables the design and constructure of a virus particle or virus hybrid with specific antigenic properties which further allows for the safe and inexpensive production of vaccines or diagnostics.
Campoletis sonorensis Virus in Heliothis virescens : and preliminary studies on the organization of the viral genome
(1986) Blissard, Gary Wilson; Summers, Max D.; Vinson, S. Bradleigh; Keeley, Larry L.; Peterson, David O.; Quarles, J. M.
The expression of Campoletis sonorensis Virus (CsV) in the parasitized host, Heliothis Virescens, was investigated. At least 12 major CsV mRNAs were detected in parasitized H. virescens larvae between 2 hours and 9 days after parasitization. Cloned CsV DNA restriction fragments which contained expressed sequences were used as hybridization probes for mapping individual CsV transcripts to superhelical regions of the CsV genome. Transcriptional mapping of several CsV mRNAs showed that the CsV genes studied are segregated on different superhelical DNAs or different sets of superhelical DNAs, thus demonstrating that the CsV genome is transcriptionally multipartite. Significant cross homology detected between CsV superhelical DNAs suggests that either (a) some CsV genes are shared on several superhelical DNAs or (b) separate superhelical DNAs may contain genes which are partially homologous. Also, the cross homology observed between different CsV superhelical DNAs was not limited to the coding regions of the genes examined in this study. Putative CsV proteins were identified by hybridization selection and in vitro translation of specific CsV mRNAs. cDNA clones homologous to a gene encoding an abundant CsV mRNA were identified. Based on Southern hybridizations of these cDNA clones to viral DNA, the cDNA clones were divided into two classes. DNA sequencing and northern blot analysis of two representative cDNA clones indicated that the two cDNA clones were synthesized from two CsV mRNAs (1.6 and 1.0 kb) which share several regions of homology. Analysis of the long open reading frame from each cDNA revealed several regions of conserved amino acid sequences although the two putative proteins have otherwise diverged in amino acid sequence. Comparison of the DNA sequence of a CsV genomic DNA restriction fragment to the sequence from a cDNA clone revealed that the CsV gene is a spliced gene which contains at least two introns. A 15.8 kbp superhelical DNA (WSp-16kA) was cloned from CsV superhelical DNA region W. The two cDNA classes were mapped to different regions of the circular map of WSp-16kA. These data show that two related CsV genes are located on a single CsV superhelical DNA molecule.
cDNA cloning and transcriptional regulation of the vitellogenin receptor from the imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae)
(Texas A&M University, 2005-02-17) Chen, Mei-Er; Keeley, Larry L.; Pietrantonio, Patricia V.; MacKenzie, Duncan S.; Summers, Max D.
Receptors that transport vitellogenin into oocytes are of vital importance to egg-laying species because they promote oocyte development. In this study, we describe the cloning of the first hymenopteran vitellogenin receptor (VgR) cDNA. Using reverse transcription polymerase chain reaction (RT-PCR) and both 5’- and 3’- rapid amplification of cDNA ends (RACE), cDNA fragments encompassing the entire coding region of a putative VgR from fire ant (= SiVgR) were cloned and sequenced. The complete SiVgR cDNA has a length of 5764 bp encoding a 1782-residue protein with a predicted molecular mass of 201.3 kDa. The deduced amino acid sequence of the SiVgR revealed that it encoded a protein belonging to the low-density lipoprotein receptor superfamily. The number and arrangement of modular domains of SiVgR are the same as those of mosquito and fruit fly VgRs, except there are only four Class A cysteine-rich repeats in the first ligand binding domain of SiVgR compared to five in the mosquito and fruit fly. The deduced amino acid sequence of the SiVgR exhibited 35% and 31% identity to those of the mosquito and fruit fly VgRs, respectively. Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in ovaries of reproductive females − both alates (virgins) and queens (mated) and was more abundant in alates. The developmental profile of transcriptional expression was determined by semiquantitative RT-PCR. It showed that the SiVgR transcript increased 6-fold from 0- to 10-days after mating, then remained constant through 30 days. It also showed that the SiVgR transcripts increased with age in alate virgin females. The transcriptional expression of the SiVgR was up-regulated more than two-fold by methoprene, a juvenile hormone analog, as determined by using an in vitro system. This suggested the SiVgR gene is JH regulated.
Chemical oxidation of tryptic digests to improve sequence coverage in peptide mass fingerprint protein identification
(Texas A&M University, 2004-09-30) Lucas, Jessica Elaine; Russell, David H.; Schweikert, Emile A.; Summers, Max D.; Vigh, Gyula
Peptide mass fingerprinting (PMF) of protein digests is a widely-accepted method for protein identification in MS-based proteomic studies. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is the technique of choice in PMF experiments. The success of protein identification in a PMF experiment is directly related to the amount of amino acid sequence coverage. In an effort to increase the amount of sequence information obtained in a MALDI PMF experiment, performic acid oxidation is performed on tryptic digests of known proteins. Performic acid was chosen as the chemical oxidant due to the ease of use and to the selective oxidation of cysteine, methionine, and tryptophan residues. In experiments performed in our laboratory, performic acid oxidation either increased or did not affect protein sequence coverage in PMF experiments when oxidized tryptic digests were analyzed by MALDI. Negative mode MALDI data were acquired, as well as positive mode MALDI data, due to the enhanced ionization of cysteic acid-containing peptides in negative mode. Furthermore, the confidence in a protein match is increased by observation of mass shifts indicative of cysteine, methionine, and/or tryptophan in oxidized peptide ion signals when comparing MALDI spectra prior to performic acid oxidation and after oxidation due to the low abundance of these residues in the majority of all known and hypothetical proteins.
Conformation and electronic configuration of complexes with multiple dimetal units
(2009-05-15) Zhao, Qinliang; Darensbourg, Marcetta Y.; Murillo, Carlos A.; Simanek, Eric E.; Summers, Max D.
By using the building blocks Mo2(DAniF)3(O2CCH3) (DAniF = N,N'-di-panisylformamidinate) and [Mo2(cis-DAniF)2(NCCH3)4](BF4)2, a series of complexes with multiple dimolybdenum units, bridged by a variety of linkers and having various oxidation states, has been synthesized and studied by various physical and chemical methods. The isomeric neutral diamidate-bridged molecules, α– and β– (DAniF)3Mo2(ArN(O)CC(O)NAr)Mo2(DAniF)3 (Ar = p-anisyl), have been oxidized to give the PF6 salts of the four cations α1+, α2+, β1+, β2+; all four structures together with supporting evidence show that in α1+ and α2+ the unpaired electrons are localized while in β1+ and β2+ they are delocalized in the time scale of these experiments. It is also found that a hydroxide bridged complex having a [Mo2](µ-OH)2[Mo2] core undergoes a oxidative deprotomation, both in solution and in crystals, to a compound with a [Mo2](µ-O)2[Mo2] core. A probable key intermediate with one OH and one O bridge has also been characterized. One electron oxidation of the tetrabridged compounds [Mo2(cis-DAniF)2]2(µ-X)4, where X is a halogen atom (Cl, Br, I), produces a decrease of about 0.24 Å in the separation between the midpoints of the multiply bonded dimolybdenum units. DFT calculations suggest partial bond formation during the oxidation, which is consistent with NIR, EPR and electrochemical measurements. Additionally, a pair of isomeric cyclic triads containing three [Mo2(cis-DAniF)2]2+ units, bridged by six fluoride anions, have been synthesized and crystallographically characterized. The symmetry of the α isomer is C2v because the three [Mo2] units are oriented in two orthogonal directions while that of the other isomer is D3h because the three [Mo2] units are parallel. No direct interconversion between isomers has been detected by heating or irradiation of solutions but oxidation of the α isomer first generates an α+ species that changes to β+. Finally, reaction of [Mo2(cis-DAniF)2(NCCH3)4](BF4)2 and Bun 4NBH4 in ether gives [Mo2(cis-DAniF)2]2(µ-H)4 (14), a compound whose Mo4H4 core may be described as an elongated tetrahedron in which the four H atoms are along the four long edges and the Mo2 units along the short edges.
Development of tandem time-of-flight instrumentation for the examination of prompt photodissociation of peptides using 193-nm radiation
(Texas A&M University, 2006-04-12) Morgan, Joseph William; Russell, David H.; Goodman, D. Wayne; Summers, Max D.; Vigh, Gyula
The design and incorporation of a decelerating/accelerating cell into a reflectron time-of-flight mass spectrometer is described for the examination of promptly-formed photodissociation products of peptide ions. The analytical utility of prompt 193-nm photodissociation was investigated for model peptides that resemble tryptic digest products, as well as for two sets of homologous peptides. The first of these sets include bradykinin, several bradykinin fragments, and two bradykinin mutants with substituted amino acids. Fragment ion spectra of [M + H]+, [M + Na]+, and [M + Cu]+ were collected for each of these peptides. The second set of homologous peptides has the sequence XVGVAZG, where variable amino acid X was either arginine, histidine, or lysine, and amino acid Z was either proline, serine, or glycine. Photofragment ion spectra obtained using the new mass spectrometer are compared to results of high energy collision induced dissociation (CID) acquired on a high performance commercial instrument. The advantages and disadvantages of prompt photodissociation relative to CID are discussed, as well as the advantages of photodissociation using the modified instrument geometry versus that of the post-source decay focusing method.
The expression and transcriptional regulatory properties of the Autographa californica nuclear polyhedrosis virus IE1 gene product
(1991) Kovacs, Gerald Raul; Summers, Max D.; Golden, James; Guarino, Linda A.; Peterson, David O.; Wild, James R.
Gene regulation in the Autographa californica nuclear polyhedrosis virus is temporally regulated and sequentially ordered. Transcription is regulated, in part, by immediate early gene products with transcriptional regulatory properties. The IE1 gene product is a transcriptional regulator of AcMNPV genes. Studies were conducted to determine its expression throughout infection, and to elucidate novel transcriptional regulatory properties. The results of S1 nuclease assays showed that IE1 and IE0 RNAs were differentially expressed. Both mRNAs were expressed during the early phase of infection; whereas, only IE1 was expressed during the late phase. Two late spliced mRNAs containing the IE0 open reading frame were also identified. Novel activities of IE1 and IE0 were identified by transient cotransfection assays. The data showed that IE1 can function as an activator and repressor of gene expression. In addition to transactivating delayed early genes (Guarino and Summers, 1986a; 1986b), IE1 stimulated its own expression. The IE1 gene product also down-regulated expression of IE0. Although IE1 and IE0 are structurally similar, they expressed different activities. Unlike IE1, IE0 did not transactivate the delayed early gene 39K, without a cis-linked hr5 enhancer element. In addition, IE0 did not show any autoregulatory properties, but it did transactivate IE1. To further studies on the mechanism of IE1-mediated gene activation, a functional dissection of the IE1 gene product was conducted. Various mutants containing amino-terminal, carboxy-terminal, and internal deletions, and site specific mutations were constructed and tested for the ability to regulate gene expression and bind to the hr5 enhancer in vitro. The results indirectly showed that the IE1 gene product has two distinct and autonomous domains that regulate transcription and DNA binding. The amino terminal 145 amino acids contains a putative acidic activation domain required for transcriptional activation and repression, and the carboxy-terminal 100 amino acids contains a putative helix-loop-helix motif that mediates DNA binding activity. Mutants that expressed only the amino terminal acidic activation domain, and lacked the DNA binding domain, specifically interfered with wild type IE1 transactivation of the delayed early reporter plasmid 39CATQ- suggesting that this region interacts with transcriptional target proteins in vivo. The IE1 gene product functions as a transcriptional regulator of viral genes, and may be essential for the proper sequential activation and repression of genes throughout a viral infection.
Identification and characterization of the IE-N gene from the Autographa californica Nuclear Polyhedrosis Virus
(1990) Carson, David Dean; Guarino, Linda A.; Summers, Max D.; Pace, Carlos N.; Park, William D.; Wilson, Van G.
To study temporal regulation of Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genes, the delayed early 39K gene was cloned from AcMNPV and linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Co-transfection with viral DNA digested with BglII suggested that a viral element was involved in the activation of the delayed early 39K promoter. This BglII-sensitive element, was localized to the PstI-N fragment of AcMNPV DNA. A 1330 nucleotide RNA spanning the BglII site was transcribed when the PstI-N fragment was transiently expressed in S. frugiperda cells. This gene encoded by the PstI-N fragment was designated IE-N. The nucleotide sequence of the IE-N gene was determined and shown to encode a serine- and glutamine-rich protein with a predicted molecular weight of 47,000. A polypeptide produced by the IE-N coding region can be detected by in vitro translation, radio-immunoprecipitation detection of an IE-NCAT fusion protein, and by protein activity. IE-N mRNA was abundantly expressed early, but not late, after infection with AcMNPV. In addition, IE-N mRNA and activity can be detected in S. frugiperda cells transiently expressing the PstI-N fragment. Because IE-N did not require the synthesis of AcMNPV gene products to be expressed, IE-N is an immediate early gene. Three viral factors regulated the transient expression of IE-N. These factors were the viral enhancer hr1, immediate early gene IE-1, and the gene product of IE-N. The hr1 stimulated expression when linked in cis to the IE-N gene. This enhancement was independent of position and orientation with respect to the IE-N gene. Co-expression of IE-1 with IE-N reduced the overall expression of IE-N. This decrease in IE-N expression by IE-1 was mediated by the hr1 enhancer sequences linked to the IE-N gene. The IE-N gene product increased expression from the IE-N gene. This stimulation was dependent upon the promoter of IE-N. In addition to increasing its own expression and that of the delayed early gene 39K, IE-N also stimulated expression of the immediate early IE-1 gene. IE-N, therefore, appears to produce a protein which functions in activating AcMNPV immediate early gene expression and possibly delayed early gene expression, as well.
Identification of the membrane association of BV/ODV E26 and the domains in BV/ODV E26 responsible for nuclear trafficking to intranuclear microvesicles
(Texas A&M University, 2007-04-25) Burks, Jared K.; Summers, Max D.; Holzenburg, Andreas; Pietrantonio, Patricia; Riley, Bruce B.
The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has two viral forms, budded virus (BV) and occlusion derived virus (ODV). The envelopment of these two viral forms occurs at different locations: BV acquires envelopes at the plasma membrane while ODV acquires envelopes in the nucleus. The two viral forms carry out different functions in the viral life cycle. The purpose of this study is to investigate how viral envelope proteins sort/traffic to the nucleus. Of particular interest is BV/ODV E26 (E26). E26 is an envelope protein of both BV and ODV (Braunagel and Summers, 1994); therefore it must traffic to the plasma membrane and the nucleus during infection. Thus, E26 is a bi-directional trafficking protein, which interacts with membranes in both locations of the cell. As such it has been shown that there are several immunoreactive forms of E26 (Beniya, Braunagel, and Summers, 1998). The da26 gene produces at least 2 protein products of 26 and 28 kDa with different functions respectively, which correlate with localization, solubility, membrane association, and temporal requirements. The 28 kDa form is likely a soluble protein that interacts with transcriptional activators and DNA in the nucleus in the early stages of infection. A part of the 26 kDa population is a membrane bound form interacting with an integral membrane protein in the ER and likely functions as an INM sorting factor. The 26 kDa membrane bond form is also found in the inner nuclear membrane, intra-nuclear microvesicles, ODV envelopes, and ODV in the nucleus.
Insect signal sequences useful to improve the efficiency of processing and secretion of foreign genes in insect systems
(United States. Patent and Trademark Office, 1992-10-13) Summers, Max D.; The Texas A&M University System
The engineering of foreign vertebrate gene constructs by recombinant DNA techniques for the more efficient processing and secretion of foreign genes in insect systems is done by replacing the foreign vertebrate protein signal peptide sequences with protein signal sequences from insect cell secreted proteins. A baculovirus expression vector system is constructed wherein the natural signal DNA peptide sequence associated with the desired foreign gene, such as CD4 (T cell surface protein T4) is replaced by the signal DNA sequence from the insect signal peptides coding for the cuticle gene or adipokinetic hormone.
Intramolecular electronic communication between dimetal units with multiple metal??al bonds
(2009-05-15) Li, Zhong; Fackler, John P.; Murillo, Carlos A.; Darensbourg, Donald J.; Gabba?Francois P.; Summers, Max D.
Lepidopteran AKH signal sequence
(United States. Patent and Trademark Office, 1991-06-11) Summers, Max D.; Bradfield, James Y.; Keeley, Larry L.; The Texas A&M University System
A baculovirus expression vector system is constructed wherein the natural signal DNA peptide sequence associated with the desired foreign gene, such as CD4 (T cell surface protein T4) is replaced by the signal DNA sequence from the Lepidopteran adipokinetic hormone (AKH) precursor signal peptides. The exemplary Manduca sexta AKH signal sequence is represented as follows: ##STR1## .
A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures.
(Texas Agricultural Experiment Station, 1987) Summers, Max D.; Smith, Gale E.
Method for producing a recombinant baculovirus expression vector
(United States. Patent and Trademark Office, 1988-05-17) Smith, Gale E.; Summers, Max D.; The Texas A&M University System
A method for producing a recombinant baculovirus expression vector, capable of expression of a selected gene in a host insect cell, is disclosed. The preferred method involves cleaving baculovirus DNA to produce a DNA fragment comprising a polyhedrin gene or portion thereof, including a polyhedrin promoter. A recombinant shuttle vector is prepared by inserting said DNA fragment into a cloning vehicle and thereafter inserting a selected gene into the thus modified cloning vehicle such that it is under the transcriptional control of the polyhedrin promoter. The recombinant shuttle vector is contacted with a baculovirus DNA so as to effect recombination and incorporation of the selected gene into the baculovirus genome.
Method for producing a recombinant baculovirus expression vector
(United States. Patent and Trademark Office, 1989-11-07) Smith, Gale E.; Summers, Max D.; The Texas A&M University System
A method for producng a recombinant baculovirus expression vector, capable of expressing a selected gene in a host insect cell, is disclosed. The method involves cleaving baculovirus DNA to produce a DNA fragment comprising a polyhedrin gene or portion thereof, including a polyhedrin promoter. A recombinant transfer vector is prepared by inserting said DNA fragment into a cloning vehicle and thereafter inserting a selected gene into the thus modified cloning vehicle such that it is under the transcriptional control of the polyhedrin promoter. The recombinant transfer vector is contacted with a baculovirus DNA so as to effect recombination and incorporation of the selected gene into the baculovirus genome. The resultant recombinant baculovirus is then used to infect susceptible insects or cultured insect cells and the protein product from the incorporated selected gene is produced from the infection.
Method to improve the efficiency of processing and secretion of foreign genes in insect systems
(United States. Patent and Trademark Office, 1994-01-11) Summers, Max D.; The Texas A&M University System
The engineering of foreign vertebrate gene constructs by recombinant DNA techniques for the more efficient processing and secretion of foreign genes in insect systems is done by replacing the foreign vertebrate protein signal peptide sequences with protein signal sequences from insect cell secreted proteins. A baculovirus expression vector system is constructed wherein the natural signal DNA peptide sequence associated with the desired foreign gene, such as CD4 (T cell surface protein T4) is replaced by the signal DNA sequence from the insect signal peptides coding for the cuticle gene or adipokinetic hormone.
Response of the Heliothis virescens (F.) larvae to nitrogen fertilization and irrigation inputs in cotton
(1988) Teague, Tina Gray; Cate, James R.; Cothren, J. Tom; Hons, Frank M.; Summers, Max D.
When high inputs of nitrogen (N) fertilizer and irrigation are used in cotton production, variation in quantity and quality of the crop as food for insect herbivores can result. Field studies were conducted in 1983-85 to investigate the effects of these inputs in cotton on survival of Heliothis virescens (F.) larvae and to provide baseline biological data for use in crop-pest simulation models. Effects of N and irrigation on larval establishment were evaluated by infesting squaring cotton with newly hatched larvae and determining survival after 7 days. Preliminary studies conducted to determine appropriate release sites for artificial infestation indicated that release on the upper 2 cm of the mainstem terminal resulted in greatest levels of larval establishment compared to release on young squares (flower buds) or vegetative leaves. When larvae were released on plants that had received varying amounts of N under irrigated and dryland conditions, larval survival generally was greatest where high rates of N were used. This response to N was most consistent under irrigated conditions. Establishment levels were lowest in older plants with maturing bolls and in cotton not contained in exclusion cages. In laboratory studies, young larvae feeding on terminal leaves from irrigated plants receiving the highest amounts of N were observed to have the greatest weight gain. Levels of leaf petiole nitrates, sugars and simple phenolics were monitored in plants used in field and laboratory studies, but relationships between these variables and larval establishment or weight gain were not clear. Response of crop plants and native populations of Heliothis and boll weevil to N fertilization and irrigation was monitored over three growing seasons on two different soil types and under chemically protected and natural crop environments. In unprotected field plots, Heliothis and boll weevil damage levels were observed at times to be increased with high N and irrigation. In protected field plots, no response by Heliothis was apparent. For both protected and unprotected studies, fertilization and irrigation resulted in continued production of squares well into the crop maturation period; this resulted in continued availability of food for boll weevil and increased boll weevil damage.
Sorting of inner nuclear membrane-directed proteins at the endoplasmic reticulum membrane
(Texas A&M University, 2006-04-12) Saksena, Suraj; Summers, Max D.; Braunagel, Sharon C.; Hu, James C.; Johnson, Arthur E.; Young, Ryland F.
The current "diffusion-retention" model for protein trafficking to the inner nuclear membrane (INM) proposes that INM proteins diffuse laterally from the membrane of the endoplasmic reticulum into the INM and are then retained in the INM by binding to nuclear proteins or DNA. Because some data indicate that the sorting of baculovirus envelope proteins to the INM is protein-mediated, we have examined the early stages of INM protein integration and sorting using photocrosslinking. Both viral and host INM-directed proteins were integrated cotranslationally through the endoplasmic reticulum translocon, and their nonrandom photocrosslinking to two translocon proteins, Sec61± and translocating chain-associated membrane protein (TRAM), revealed that the first transmembrane sequence (TMS) of each viral and host INM-directed protein occupied a very similar location within the translocon. Because few TMSs of non-INM-directed membrane proteins photocrosslink to TRAM, it seems that the INM-directed TMSs occupy different sites within the translocon than do non-INM-directed TMSs. The distinct proximities of translocon components to INM-directed TMSs strongly suggest that such TMSs are recognized and initially sorted within the translocon. Previous work with the envelope protein ODV-E66 (E66) showed that E66 trafficking to the INM is mediated via an INM sorting signal (Sorting Motif or SM). In this study, using a site-specific crosslinking approach we demonstrate that following ER membrane integration, the SM is adjacent to two viral proteins: FP25K & BV/ODV-E26 (E26). Deletion of FP25K from the viral genome results in the accumulation of E66 at the ONM, suggesting that FP25K may facilitate protein movement at the nuclear pore. While the role of the E66-E26 interaction remains to be determined, these data suggest that E66 trafficking to the INM is a protein-facilitated process. Crosslinking experiments using E66 integration intermediates revealed that during co-translational integration at the ER, the SM is adjacent to two cellular proteins of ~10kDa and ~25kDa, referred to as SMAP 10 (SM associated protein of 10kDa) & SMAP25 respectively. Thus, contrary to the widely accepted "diffusion-retention" model for protein trafficking to the INM, our data indicate that protein sorting to the INM is a multistep process initiated upon membrane integration in which the INM sorting signal sequentially associates with various sorting factors.
Trafficking of integral membrane proteins of the inner nuclear membrane can be mediated by the ''sorting motif'' of autographa californica nucleopolyhedrovirus odv-e66
(Texas A&M University, 2006-10-30) Williamson, Shawn T; Summers, Max D.; Braunagel, Sharon C.; Johnson, Arthur E.; Lekven, Arne C.; Siegele, Deborah A.
The amino-terminal 33 amino acids of the baculovirus integral membrane protein, ODV-E66, are sufficient for localization of fusion proteins to viralinduced intranuclear microvesicles (MV) and occlusion derived virus envelopes during infection, and has been termed the sorting motif (SM). When abundantly expressed, SM-fusions are also detected in the inner nuclear membrane (INM), outer nuclear membrane and endoplasmic reticulum of infected cells, suggesting proteins with the SM use the same trafficking pathway as cellular INM proteins to traffic to nuclear membranes. This study identifies the essential characteristics required for sorting of the SM to the INM of uninfected cells, and the MV and ODV envelopes of infected cells. These features are an 18 amino acid transmembrane sequence that lacks polar and charged amino acids (a.a.) with a cluster of charged a.a. spaced 5-11 residues from the end of the transmembrane sequence. A comparison of the a.a. sequence of these SM features with cellular INM proteins shows the features are conserved. The model of INM protein sorting and localization predicts the only known sorting event during INM protein trafficking is immobilization/retention in the INM. This study uses confocal microscopy and fluorescence recovery after photobleaching to compare the localization and mobility of lamin B receptor (LBR) fusions (which contain SM-like sequences) to a viral SM fusion when expressed in either mammalian or insect cells. The results show that immobilization is not necessarily required for accumulation of proteins in the INM. Furthermore, the results from infected cells show that an active sorting event, likely independent of immobilization, can distinguish the viral SM from cellular sequences similar to the SM. The results of this study show that sorting of proteins to the INM can be mediated by the viral SM or INM protein SM-like sequences that can function either independent of, or in addition to, immobilization. These data combined with recent reports suggest that in addition to diffusion:retention a signal mediated mechanism for sorting and localization to the INM can occur.
Unique nucleotide and amino acid sequence and uses thereof
(United States. Patent and Trademark Office, 2000-01-25) Summers, Max D.; Braunagel, Sharon C.; Hong, Tao; The Texas A & M University System
Provided are hydrophobic targeting sequences, which may serve to target heterologous proteins to a variety of cellular membranes. In particular, the structural components of the nuclear envelope, or those components which become nucleus-associated, may be targeted with the sequences provided. Also provided are methods of targeting heterologous proteins to particular membranes, and the use of these targeted proteins in therapeutic, diagnostic and insecticidal applications.

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