Metal-regulated transporters and uses therefor

Details Metal-regulated transporters and uses therefor Metal-regulated transporters and uses therefor

2000-05-25

2003-07-08

Bui, Phuong T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C424S058000, C424S195110, C435S183000, C435S410000, C435S419000, C435S320100, C530S350000, C530S370000, C536S023100, C536S023200, C536S023600, C800S295000

Reexamination Certificate

active

06590140

ABSTRACT:

BACKGROUND OF THE INVENTION
Iron deficiency is one of the most common human nutritional disorders in the world today (Yip, R. (1994)
J. Nutr
. 124: 1479S-1490S). Indeed, iron is an essential nutrient for virtually all organisms because it plays a critical role in important biochemical processes such as respiration and photosynthesis. Although abundant in nature, iron is often available in limited amounts because the oxidized form, Fe(III), is extremely insoluble at neutral or basic pH. This fact is of particular importance to agriculture because approximately one-third of the world's soils are classified as iron-deficient (Yi, Y. et al. (1994)
Plant Physiol
. 104: 815-820). Many “iron-efficient” plant varieties have iron uptake strategies (designated strategy I or strategy II) that, not surprisingly, are directed at solubilizing iron (Römheld, V. (1987)
Physiol. Plant
. 70: 231-234). Strategy II plants, which include all of the grasses, release Fe(III) compounds called “phytosiderophores” into the surrounding soil that bind iron and are then taken up into the roots. Most other iron-efficient plants use strategy I and respond to iron deprivation by inducing the activity of membrane-bound Fe(III) chelate reductases that reduce Fe(III) to the more soluble Fe(II) form. The Fe(II) product is then taken up into the roots by an Fe(II) specific transport system that is also induced by iron-limiting growth conditions. Furthermore, the roots or strategy I plants release more protons when iron-deficient, lowering the rhizosphere pH and thereby increasing the solubility of Fe(III). Thus, it would be desirable to take advantage of this understanding of iron-uptake strategies to produce plants which have increased iron-uptake capabilities.
Furthermore, another metal, zinc, is an integral cofactor of many proteins and is indispensable to their catalytic activity and/or structural stability (Vallee and Auld (1990)
Biochemistry
9:5647-5659). Moreover, zinc is a ubiquitous component of enzymes involved in transcription and of accessory transcription factors, the zinc finger proteins, that regulate gene expression (Rhodes and Klug (1993)
Sci. Am
. 268(2):56-65). Because of the many roles this metal plays in cellular biochemistry, zinc is an essential nutrient for all organisms. Despite this importance, very little is known about the molecular mechanisms cells use to obtain zinc. No transporter genes involved in zinc uptake (i.e. influx transporters) have been isolated from any organism. Recently, genes have been identified whose products are responsible for detoxifying intracellular zinc by transporting the metal from the cytoplasm to the cell exterior or into intracellular compartments (i.e. efflux transporters). These genes include the closely related eukaryotic genes, COT1, ZRC1, and Znt-1 (Conklin et al. (1992)
Mol. Cell Biol
. 12:3678-3688; Kamizono et al. (1989)
Mol. Gen. Genet
. 219:161-167; Palmiter and Findley (1995)
EMBO J
. 14:639-649). While important for zinc detoxification, these genes do not appear to play a role in zinc uptake.
In addition, metal ion pollution is perhaps one of the most difficult environmental problems facing the industrial world today. Unlike the organic and even halogenated organic pollutants, which can be degraded in the soil, metals are essentially nonmutable. The electrolytic, in situ immobilization and chemical leaching technologies for cleaning polluted sites are all very expensive, particularly in light of how vast some of these sites are. With the exception of approaches like vitrification, most in situ metal ion remediation schemes require some mechanism for increased mobilization of the metal ion. This raises the possibility of further endangering local wildlife or adjacent ecosystems not already affected. Thus, a need still exists for better methods for removing toxic pollutants from the soil.
Accordingly, an object of the invention is to generate transgenic plants in which expression of an MRT polypeptide is altered such that metal-uptake is increased.
Another object of the invention to provide methods for removing toxic pollutants, such as heavy metals, from the environment.
Yet another object of the invention is to provide methods for improving human or animal nutrition, e.g., for treating metal-deficiency, e.g., iron-deficiency or zinc-deficiency.
SUMMARY OF THE INVENTION
This invention is based, at least in part, on the discovery of a family of polypeptides, designated herein as metal-regulated transporter, MRT, polypeptides, which share several structural/functional properties, at least one of which is related to metal transport. Structurally, the MRT polypeptides include, for example, at least one transmembrane binding domain which has at least about 40%, more preferably at least about 50%, 55%, 60%, 70%, 80% or 90% amino acid sequence identity with an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14 and/or at least one histidine rich domain. Functionally, the MRT polypeptides are capable of, for example, transporting metals, e.g., Fe, e.g., Fe(II), Cd, Co, Mn, Pb, Hg and/or Zn.
Preferred MRT polypeptides have an overall amino acid sequence identity of at least about 40%, preferably at least about 42%, 45%, 47%, 50%, more preferably at least about 55%, 60%, 70%, 80%, 90%, or 95% with an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14; it has eight transmembrane domains; it has four histidine rich domains; or it can be isolated from the Arabidopsis family of plants.
Accordingly, this invention pertains to isolated nucleic acid molecules encoding an MRT polypeptide. Such nucleic acid molecules (e.g., cDNAs) have a nucleotide sequence encoding an MRT polypeptide (e.g., an
A. thaliana
IRT1 polypeptide, an
A. thaliana
IRT2 polypeptide, an
A. thaliana
ZIP1 polypeptide, an
A. thaliana
ZIP2 polypeptide, or an
A. thaliana
ZIP3 polypeptide) or biologically active portions or fragments thereof, such as a polypeptide having an MRT bioactivity. In a preferred embodiment, the isolated nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:13, or a portion or fragment thereof. Preferred regions of these nucteotide sequences are the coding regions. Other preferred nucleic acid molecules are those which have at least about 45%, preferably at least about 48%, more preferably at least about 50%, and most preferably at least about 55%, 60%, 70%, 80%, 90%, 95%, 97% or 98% or more nucleotide sequence identity over the entire sequence with a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:13, or a portion or fragment thereof. Nucleic acid molecules which hybridize under stringent conditions to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:13, e.g., nucleic acid molecules which hybridize to at least 6 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:13, are also within the scope of the invention. Such portions or fragments include nucleotide sequences which encode, for example, polypeptide domains having an MRT bioactivity. Examples of portions or fragments of nucleic acid molecules which encode such domains include portions or fragments of nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:13 which encode one or more of the following: at least one transmembrane domain which has at least about 40%, more preferably at least about 50%, 55%, 60%, 70%, 80% or 90% amino acid sequence identity with an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14 or at least one histidine rich domain. Nucleic acid molecules of the present invention which further comprise a label are also within the scope of the invention. Complements of the nucleic acid molecules of the present invention are also specifically contemplated.
In another embodiment, the nucleic aci

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